Epidemics of tick-borne encephalitis involving thousands of humans occur annually in the forested regions of Europe and Asia. Despite the importance of this disease, the underlying basis for the development of encephalitis remains undefined. Here, we prove the key role of CD8(+) T-cells in the immunopathology of tick-borne encephalitis, as demonstrated by prolonged survival of SCID or CD8(-/-) mice, following infection, when compared with immunocompetent mice or mice with adoptively transferred CD8(+) T-cells. The results imply that tick-borne encephalitis is an immunopathological disease and that the inflammatory reaction significantly contributes to the fatal outcome of the infection.

Sera obtained at enrollment in the study from patients suffering from moderate to sever dysplasia (cervical intraepithelial neoplasia grade II), carcinoma in situ (cervical intraepithelial neoplasia grade III) and invasive carcinoma, or developing any of these conditions in the course of the prospective study, and from control subjects, were examined for herpes simplex type-2 (HSV-2) antibody presence. The controls were matched with the patients by age, age at first intercourse, number of sexual partners, smoking habits and history of diathermoelectrocoagulation of the ectopic epithelium and transformation zone of cervix. Only those subjects were selected as controls who remained free of pathological colposcopical and cytological findings throughout the observation period, i.e. for at least 4 years after their serum sample was obtained. The microneutralization test (MNT) and type-2-specific solid-phase radioimmunoassay (SPRIA) were used as serological tests. No difference in the prevalence of HSV-2 antibody between the patients and controls was revealed by either test. Various combinations of the results from the two tests also failed to show any difference between patients and controls. Moreover, no significant differences were observed in the prevalence of HSV-2 antibody between patients suffering from the various pathological conditions and those diagnosed at enrollment and later in the course of the study. These results do not provide any support for the hypothesis of the involvement of HSV-2 in cervical neoplasia.

To determine the risk associated with previous herpes simplex type-2 (HSV-2) infection and possibly other virus infections, a prospective study of cervical neoplasia in more than 10,000 women was performed in the 1975-1983 period. The subjects were selected at random from an alphabetical listing of eligible women living in one district of Prague. At enrollment colposcopy and cervical cytology were performed, a blood sample was taken and data regarding education, socio-economic status, personal habits and sexual and reproduction-associated attributes were obtained from each woman. A total of 10,683 women were enrolled; a complete set of data was obtained in 10,389 women. Women with normal or non-significant findings were invited for further colposcopical and cytological investigations after 2 years and 4 years, the other women were followed at 3- to 6-monthly intervals. In women with highly significant findings, histological investigation was performed. The total of 150 cases of moderate to severe dysplasia (i.e. cervical intraepithelial neoplasia, grade II, CIN II), 83 cases of carcinoma in situ (CIN III) and 21 cases of invasive carcinoma (INCA) were detected. More than 60% of the patients were ill at enrollment, the other cases developed in subjects with originally slightly suspicious (27 CIN II, 17 CIN III, 3 INCA) or negative findings (30 CIN II, 12 CIN III, 3 INCA). Analysis of the data indicated significantly positive correlation of one or more of these clinical conditions with a number of sexual and reproduction-related attributes of which early age at first intercourse was most consistent. Among the other attributes, the smoking habit was associated with the highest risk of developing the disease. A negative correlation of cervical neoplasia with several attributes was demonstrated; of these diathermoelectrocoagulation of the ectopic epithelium and transformation zone of cervix was the most important single protective factor. On the basis of these findings, control subjects were selected for serological studies.

The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC ؊ toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8 ؉ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaA⌬AC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8؉ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b ؉ target cells and pave the way for the construction of CyaA⌬AC-based polyvalent immunotherapeutic T cell vaccines.

Abstract. Experimental activation of peritoneal macrophages by interferon gamma (IFN-γ) resulted in the inhibition of Encephalitozoon cuniculi replication. However, E. cuniculi could replicate either in a non-activated cell line of murine macrophages PMJ2-R or in IFN-γ-activated PMJ2-R cells. Moreover, activation with IFN-γ led to faster replication of E. cuniculi in these cells. Opsonisation of E. cuniculi spores with anti-E. cuniculi polyclonal antibody did not affect E. cuniculi replication in both, non-activated and activated murine macrophages. In contrast, opsonisation of E. cuniculi spores caused the most effective replication of E. cuniculi in activated PMJ2-R cells. However, production of nitric oxide by these cells was significantly more intensive than that in non-activated, infected cells, where the parasite replicated to a much lesser extent. Our results support the hypothesis that E. cuniculi uses phagocytosis for the infection of host cells. They also indicate that the mechanism by which spores of E. cuniculi are killed by macrophages is not dependent on nitric oxide and they reveal that PMJ2-R cells cannot substitute peritoneal murine macrophages in immunological studies on E. cuniculi.

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Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC À ) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC À constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of c-IFN-secreting CMV-specific CD8 þ T cells, as well as increase of total IFN-c and TNF-a production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC À constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC À constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

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