Korostoff, Johnathan M

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  • Publication
    Differential Effect of the Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans on Co-Cultures of Human Oral Cells
    (2005-08-01) Korostoff, Johnathan M; Kang, Philip; Volgina, Alla; DiRienzo, Joseph M; Grzesik, Wojciech
    The periodontal pathogen Actinobacillus actinomycetemcomitans expresses a cytolethal distending toxin (CDT) that typically arrests the growth of eukaryotic cells at either the G0/G1 or G2/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 post-intoxication. Epithelial cells and HPLFs were detected and counted in co-cultures following cell-specific 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.
  • Publication
    Neonatal Exposure to Thymotropic Gross Murine Leukemia Virus Induces Virus-Specific Immunologic Nonresponsiveness
    (1990-12-01) Korostoff, Johnathan M; Nakada, Marian T; Faas, Susan J; Gaulton, Glen N; Blank, Kenneth J
    Neonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific 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 of neonatally 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 of immunologic nonresponsiveness to viruses.
  • Publication
    Evaluation of the Humoral Immune Response to the Cytolethal Distending Toxin of Aggregatibacter Actinomycetemcomitans Y4 in Subjects With Localized Aggressive Periodontitis
    (2009-04-01) Xynogala, Ioanna; DiRienzo, Joseph M; Volgina, Alla; Korostoff, Johnathan M
    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. Methods:  Ninety-five subjects provided a total of 692 serum samples. Sera were analysed by enzyme-linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony-forming assay using Chinese hamster ovary cells. Results:  Fourteen of the 95 subjects exhibited significant serum Cdt-binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt-mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20–75%). Conclusions:  Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti-Cdt response may in part explain their relative susceptibility to the disease.
  • Publication
    Functional and Structural Characterization of Chimeras of a Bacterial Genotoxin and Human Type I DNAse
    (2009-02-01) DiRienzo, Joseph M; Cao, Linsen; Volgina, Alla; Korostoff, Johnathan M; Bandelac, Georges
    Chimeras composed of the cdtB gene of a novel bacterial genotoxin and the human type I DNAse I gene were constructed and their products characterized relative to the biochemical and enzymatic properties of the native proteins. The product of a cdtB/DNAse I chimera formed a heterotrimer with the CdtA and CdtC subunits of the genotoxin, and targeted mutations increased the specific activity of the hybrid protein. Expression of active chimeric gene products established that the CdtB protein is an atypical divalent cation-dependent endonuclease and demonstrated the potential for genetically engineering a new class of therapeutic agent for inhibiting the proliferation of cancer cells.
  • Publication
    Characterization of Point Mutations in the cdtA Gene of the Cytolethal Distending Toxin of Actinobacillus Actinomycetemcomitans
    (2005-12-01) Cao, Linsen; Volgina, Alla; Korostoff, Johnathan M; Huang, Chuang-ming; DiRienzo, Joseph M
    The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 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 enzyme-linked 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.
  • Publication
    Molecular and functional heterogeneity and identification of fibroblasts
    (2024-06-28) Ko, Annette; Sahingur, Sinem Esra; Chang, Yu-Cheng; Ko, Kang I; Korostoff, Johnathan M; Chang, Yu-Cheng
    Introduction: Oral wounds heal with exceptional regenerative capacity, which contrasts skin wound repair. Paired related homeobox 1 (Prrx1) is a marker for mesenchymal progenitors in bone, and recent studies have demonstrated presence and functional significance of Prrx1+ fibroblasts in dermis. Our previous findings suggest pro-healing role of Prrx1+ oral fibroblasts in mouse model, thus we sought to examine equivalent Prrx1+ cells using human gingival tissue biopsies. Here, we hypothesize that human Prrx1+ fibroblasts reside in different oral gingival tissues and that they exhibit enhanced regenerative potential for wound healing. Methods: Human gingival samples were collected from the anterior rugae, healed sites (distal wedge, edentulous crest), and tooth-associated sites (marginal gingiva from crown lengthening procedure or osseous surgery) in accordance to approved IRB protocol. The tissues were processed in paraffin-embedded block and sectioned at 5um. Immunofluorescence staining was performed with antibodies against Prrx1 and vimentin to detect Prrx1+ oral fibroblasts in various oral anatomic sites. Image analysis was carried out by counting spindle-shaped vimentin+ and Prrx1+ cells in lamina propria, normalized by area (mm2). To examine the regenerative potential of Prrx1+ cells in oral tissues, human anterior rugae samples were collected and processed for single-cell RNA sequencing (scRNA-seq). Transcriptomic analysis was performed after combining with the public scRNA-seq data (GEO: GSE164241, GSE152042). Results: Prrx1+ fibroblasts were present throughout all types of oral soft tissues. Anterior palate tissue had 72% fibroblasts expressing Prrx1, which was significantly more than those found in distal wedge, crestal tissue and marginal gingivae (39%, 30%, 26%, respectively; P<0.05) in the reticular layer of lamina propria and submucosa. ScRNA-seq analysis of the combined data demonstrated upregulation of several WNT-associated genes in Prx1+ fibroblasts, such as SFRP1 and SLPI, which had previously been reported to be involved in stem cell function and regeneration. The transcriptomic analysis further revealed that Prx1+ fibroblasts exhibit progenitor phenotype with projected differentiation trajectory toward chemotactic fibroblasts and expedited wound healing potential via immunomodulation in innate immune response. Further characterization of these inflammatory fibroblasts showed high expression of intercellular adhesion molecule-1 (ICAM-1), suggesting its potential usage as a surface marker for identification. Conclusion: Our results demonstrate that human Prrx1+ oral fibroblasts reside in the deep layer of the lamina propria, particularly in the anterior palate at higher frequency compared to other anatomic sites. These fibroblasts may exhibit improved regenerative potential via modulating WNT-associated pathway and rapid resolution of inflammation. The findings provide new insight into the enhanced healing potential of oral fibroblast subpopulations that may contribute to developing surgical techniques targeted to improve unpredictable soft tissue augmentation.