SummaryThe Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G 0 /G 1 or G2/ M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA , cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzymelinked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5 ′ ′ ′ ′ -and 3 ′ ′ ′ ′ -ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro . The mutations in mutA81 and mutA221 disrupted holotoxin formation.The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function.

Resistance to treatment and the appearance of secondary tumors in head and neck squamous cell carcinomas (HNSCC) have been attributed to the presence of cells with stem-cell-like properties in the basal layer of the epithelium at the site of the lesion. In this study, we tested the hypothesis that these putative cancer stem cells (CSC) in HNSCC could be specifically targeted and inhibited. We found that 9 of 10 head and neck tumor biopsies contained a subpopulation of cells that expressed CD133, an unusual surface-exposed membrane-spanning glycoprotein associated with CSC. A genetically modified cytolethal distending toxin (Cdt), from the periodontal pathogen Aggregatibacter actinomycetemcomitans, was conjugated to an anti-human CD133 monoclonal antibody (MAb). The Cdt-MAb complex preferentially inhibited the proliferation of CD133 + cells in cultures of established cell lines derived from HNSCC. Inhibition of the CD133 + cells was rate-and dosedependent. Saturation kinetics indicated that the response to the Cdt-MAb complex was specific. Healthy primary gingival epithelial cells that are native targets of the wild-type Cdt were not affected. Analysis of these data provides a foundation for the future development of new therapies to target CSC in the early treatment of HNSCC. Abbreviations: Cdt, cytolethal distending toxin; CSC, cancer stem cells; HNSCC, head and neck squamous cell carcinoma; MAb, monoclonal antibody.

Introduction-Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt-specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin-specific antibody response in a cohort of LAP patients and age-, race-and sex-matched controls.

SummaryNeonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virusspecific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness . The specificity of this effect at the levels of both T and B cells was demonstrated by the ability ofneonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction ofimmunologic nonresponsiveness to viruses. mmunologic nonresponsiveness to autologous molecules and a variety of exogenous antigens can be linked to their presence during neonatal maturation (1-4). The consequences of neonatal exposure to viruses are considerably more variable (5, 6). Mice infected either neonatally or congenitally with lymphocytic choriomeningitis virus (LCMV)' or endogenous ecotropic retroviruses mount potent Ig responses and produce functional CTL precursors (7-9) . However, several recent studies have shown that exposure of neonatal mice to Moloney murine leukemia virus significantly reduced the frequency ofMoloney virus-specific CTL precursors and Ig levels (6, 10) . These contrasting results raise important questions concerning the ability of different viruses to induce tolerance, and the contributions of T and B cell components to virus-specific tolerance . To address these questions, the immune response ofmice exposed as neonates to NB-tropic Gross murine leukemia virus (GMuLV) was investigated . Exposure of neonates to GMuLV induces a state ofimmunologic nonresponsiveness that may be linked to intrathymic replication of this retrovirus .1 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; GMuLV, Gross murine leukemia virus ; HAU, hemagglutinating units; LCMV, lymphocytic choriomeningitis virus; PFU, plaque-forming units ; RT, reverse transcriptase.Intraperitoneal injection of neonatal mice with GMuLV leads to the development of lymphatic leukemias typified by the presence of thymic lymphomas, and to a unique form ofpersistent infection within the central nervous system white matter (11-13) . The data reported here demonstrate that neonatal exposure to these murine retroviruses also induced a state of virus-specific T cell nonresponsiveness, characterized by impaired delayed type hypersensitivity and in vitro proliferative responses. Concomitant with this T cell defect was the functional inactivation of virus-specific B cells, as evidenced by the absence ofbot...

The periodontal pathogen Actinobacillus actinomycetemcomitans expresses a cytolethal distending toxin (CDT) that typically arrests the growth of eukaryotic cells at either the G 0 /G 1 or G 2 /M phase of the cell cycle. It was previously found that CDT failed to arrest the growth of human periodontal ligament fibroblasts (HPLFs) when grown in pure culture. In contrast, proliferation of an oral epithelial cell line was rapidly inhibited by the toxin. In this study, the feasibility of using mixed-cell cultures and cell-specific markers to evaluate the response of oral cells, when in heterogeneous populations, to CDT was established. Proliferation of epithelial cells was rapidly inhibited and the cells were selectively eliminated in co-culture with HPLFs or cementoblasts by 24-48 h postintoxication. Epithelial cells and HPLFs were detected and counted in co-cultures following cellspecific immunolabelling with antibodies against simian virus 40 large T antigen and the Ab-1 surface antigen, respectively. These results demonstrated that the activities of potential virulence factors, such as CDT, from periodontal pathogens can be successfully examined in mixed-cell cultures. This approach is especially relevant to infectious diseases that affect tissues with a diverse cellular composition, such as the periodontium.