Photobiomodulation of Gingival Cells Challenged with Viable Oral Microbes.

The oral cavity, a unique ecosystem harboring diverse microorganisms, maintains health through a balanced microflora. Disruption may lead to disease, emphasizing the protective role of gingival epithelial cells (GECs) in preventing harm from pathogenic oral microbes. Shifting GECs' response from proinflammatory to antimicrobial could be a novel strategy for periodontitis. Photobiomodulation therapy (PBMT), a nonpharmacologic host modulatory approach, is considered an alternative to drugs. While the host cell response induced by a single type of pathogen-associated molecular patterns (PAMPs) was widely studied, this model does not address the cellular response to intact microbes that exhibit multiple PAMPs that might modulate the response. Inspired by this, we developed an in vitro model that simulates direct interactions between host cells and intact pathogens and evaluated the effect of PBMT on the response of human gingival keratinocytes (HGKs) to challenge viable oral microbes at both the cellular and molecular levels. Our data demonstrated that LED pretreatment on microbially challenged HGKs with specific continuous wavelengths (red: 615 nm; near-infrared: 880 nm) induced the production of various antimicrobial peptides, enhanced cell viability and proliferation, promoted reactive oxygen species scavenging, and down-modulated proinflammatory activity. The data also suggest a potential explanation regarding the superior efficacy of near-infrared light treatment compared with red light in enhancing antimicrobial activity and reducing cellular inflammation of HGKs. Taken together, the findings suggest that PBMT enhances the overall barrier function of gingival epithelium while minimizing inflammation-mediated breakdown of the underlying structures.

Variants of Porphyromonas gingivalis lipopolysaccharide alter lipidation of autophagic protein, microtubule-associated protein 1 light chain 3, LC3.

Porphyromonas gingivalis often subverts host cell autophagic processes for its own survival. Our previous studies document the association of the cargo sorting protein, melanoregulin (MREG), with its binding partner, the autophagic protein, microtubule-associated protein 1 light chain 3 (LC3) in macrophages incubated with P. gingivalis (strain 33277). Differences in the lipid A moiety of lipopolysaccharide (LPS) affect the virulence of P. gingivalis; penta-acylated LPS1690 is a weak Toll-like receptor 4 agonist compared with Escherichia coli LPS, whereas tetra-acylated LPS1435/1449 acts as an LPS1690 antagonist. To determine how P. gingivalis LPS1690 affects autophagy we assessed LC3-dependent and MREG-dependent processes in green fluorescent protein (GFP)-LC3-expressing Saos-2 cells. LPS1690 stimulated the formation of very large LC3-positive vacuoles and MREG puncta. This LPS1690 -mediated LC3 lipidation decreased in the presence of LPS1435/1449 . When Saos-2 cells were incubated with P. gingivalis the bacteria internalized but did not traffic to GFP-LC3-positive structures. Nevertheless, increases in LC3 lipidation and MREG puncta were observed. Collectively, these results suggest that P. gingivalis internalization is not necessary for LC3 lipidation. Primary human gingival epithelial cells isolated from patients with periodontitis showed both LC3II and MREG puncta whereas cells from disease-free individuals exhibited little co-localization of these two proteins. These results suggest that the prevalence of a particular LPS moiety may modulate the degradative capacity of host cells, so influencing bacterial survival.

Cell junction remodeling in gingival tissue exposed to a microbial toxin.

UNLABELLED: The gingival epithelium plays a key role in protecting the supporting structures of the teeth from bacteria and their products. In ex vivo experiments, we recently showed that the cytolethal distending toxin (Cdt) from the periodontal pathogen Aggregatibacter actinomycetemcomitans causes extensive damage to gingival tissue. Morphological changes included detachment of the keratinized outer layer, distention of spinous and basal cells in the oral epithelium, disruption of rete pegs, and apparent dissolution of cell junctions. Adherens junctions (zonula adherens) are essential for maintaining barrier function and integrity of gingival epithelium. Therefore, immunohistochemical and RT-PCR analyses of human gingival explants (HGX) and human gingival epithelial cells (HGEC) were utilized for a closer examination of the effects of the Cdt on E-cadherin, the key membrane component of adherens junctions. Although there was some variability among tissue donors, exposure of gingival tissue or isolated epithelial cells to the toxin generally resulted in a pronounced increase in the expression and cytosolic distribution of E-cadherin, accompanied by an increase in levels of the intracellular scaffolding proteins ß-catenin and ß-actin. These results indicate that the Cdt induced substantial remodeling of adherens junctions, with a potential impact on the barrier function of gingival epithelium. ABBREVIATIONS: cytolethal distending toxin (Cdt), 4',6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), transepithelial resistance (TER).

Cytolethal distending toxin damages the oral epithelium of gingival explants.

UNLABELLED: The cytolethal distending toxin (Cdt), expressed by the periodontal pathogen Aggregatibacter actinomycetemcomitans, inhibits the proliferation of cultured epithelial cells by arresting the cell cycle. The gingival epithelium is an early line of defense against microbial assault. When damaged, bacteria collectively gain entry into underlying connective tissue where microbial products can affect infiltrating inflammatory cells, leading to the destruction of the attachment apparatus. Histological evaluation of rat and healthy human gingival tissue exposed ex vivo to the Cdt for 36 and 18 hours, respectively, revealed extensive detachment of the keratinized outer layer and distention of spinous and basal cells in the oral epithelium. Treated human tissue also exhibited disruption of rete pegs and dissolution of cell junctions. Cells in the connective tissue appeared unaffected. Primary gingival epithelial cells, but not gingival fibroblasts, isolated from the same healthy human tissue were cell-cycle-arrested when treated with the toxin. These findings provide new evidence that the Cdt severely damages the oral epithelium, ex vivo, by specifically targeting epithelial cells, in situ. The Cdt shows preferential targeting of the epithelium as opposed to connective tissue in animal and human gingival explant models. ABBREVIATIONS: cytolethal distending toxin (Cdt), connective tissue (CT), 4',6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), junctional epithelium (JE), oral epithelium (OE), rete pegs (RP), sulcular epithelium (SE).

Targeted inhibition of CD133+ cells in oral cancer cell lines.

UNLABELLED: 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 dose-dependent. 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.

Evaluation of the humoral immune response to the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans Y4 in subjects with localized aggressive periodontitis.

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.

Heparan sulfate interacting protein (HIP/L29) negatively regulates growth responses to basic fibroblast growth factor in gingival fibroblasts.

Basic fibroblast growth factor (bFGF) modulates gingival growth, and its release from heparan sulfate (HS) in the extracellular matrix (ECM) governs local tissue bioavailability. We identified a heparin/HS interacting protein (HIP/L29) that recognizes specific HS sequences. We hypothesize that HIP/L29, by modulating the interactions of bFGF with HS chains on proteoglycans, could regulate bFGF bioavailability. To investigate interactions between bFGF and HIP/L29, we isolated and cultured fibroblasts from normal gingiva and overgrown gingiva from patients on cyclosporine (CSA). bFGF significantly stimulated gingival fibroblast proliferation with or without heparin. Recombinant human HIP/L29 dramatically decreased bFGF-induced proliferation, but did not alter responses to insulin-like growth factor-1 (IGF-1). Analysis of mitogen-activated protein kinase (MAPK) phosphorylation patterns showed that bFGF stimulation of p44 (Erk-1), but not p42 (Erk-2), also was inhibited by HIP/L29 in a dose-dependent manner. Together, these results support our hypothesis that HIP/L29 modulates the bioavailability and action of bFGF.

Maintenance of oxidative phosphorylation protects cells from Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis.

Subnanomolar concentrations (3 x 10(-10) M) of Actinobacillus actinomycetemcomitans leukotoxin (Ltx) trigger apoptosis of JY cells, as shown by a decrease in mitochondrial transmembrane potential (DeltaPsim), hyperproduction of reactive oxygen species (ROS) and release of cytochrome c from the intermembrane space. When compared with heat-inactivated leukotoxin (DeltaI Ltx) controls, ATP levels in Ltx-treated JY cells continued to decrease during a 24 h experiment while cytoplasmic ADP concentrations were increasing. These results suggest that a blockage occurred in ATP/ADP exchange. To maintain ATP/ADP exchange, JY cells were transfected with bcl-2 and bcl-xL and incubated with Ltx. ATP levels of the transfected cells decreased to 67% (JY/bcl-2) and 73% (JY/bcl-xL) after the experiment. Furthermore, cytochrome c remained localized to the mitochondrial fraction of Ltx-treated JY/bcl-2 and JY/bcl-xL cells, whereas its presence in the cytoplasmic fraction of JY/gen cells suggests an uncoupling of electron transport. Expression of bcl-2 and bcl-xL in cells inhibited downstream apoptotic events such as cleavage of poly(ADP-ribose) polymerase, DNA fragmentation and activation of a family of caspases. The results indicate that Ltx induces apoptosis through a mitochondrial pathway that involves decreased levels of the ADP in the mitochondrial matrix, a lack of substrate for ATP synthetase and arrest of oxidative phosphorylation.

Beyond the specific plaque hypothesis: are highly leukotoxic strains of Actinobacillus actinomycetemcomitans a paradigm for periodontal pathogenesis?

Actinobacillus actinomycetemcomitans is a facultative anaerobe implicated in a variety of periodontal diseases. Its presence is most closely associated with localized juvenile periodontitis (LIP), although the exact role of the organism in this and other periodontal diseases is not entirely clear. While A. actinomycetemcomitans produces several different putative virulence factors, the most widely studied is the leukotoxin. The leukotoxin selectively kills polymorphonuclear leukocytes and macrophages in vitro, constituting the host's first line of defense. Interestingly, even though all strains of A. actinomycetemcomitans have the genes encoding the leukotoxin, there is variability in leukotoxin expression. Differences in the structure of the promoter region of the leukotoxin gene operon were shown to correlate directly with levels of leukotoxin production. Highly leukotoxic forms appear to exhibit increased pathogenic potential, as evidenced by recent studies that have shown a significant association between the prevalence of such strains and the occurrence of LIP in several different populations. This represents the first demonstration of an association between a particular subset of a pathogenic species and a specific periodontal disease. Early identification of A. actinomycetemcomitans by microbial and genetic assays to evaluate leukotoxicity may enhance the efficacy of preventive and/or therapeutic techniques. Future investigations should continue to evaluate pathogenic variations of additional virulence factors expressed in vivo, not only of A. actinomycetemcomitans, but also of other periodontal bacteria and infectious disease pathogens.

A multidisciplinary approach to the diagnosis and treatment of early-onset periodontitis: a case report.

BACKGROUND: The diagnosis and treatment of early-onset forms of periodontitis (EOP) represent a major challenge to periodontists. In this case report, we describe a multidisciplinary approach for the treatment of a patient with severe generalized juvenile periodontitis (GJP). Our approach incorporates clinical laboratory evaluation with conventional concepts of periodontal pathogenesis and therapeutics to diagnose and effectively treat EOP. METHODS: The 17-year-old female patient presented with clinical and radiographic evidence of severe attachment loss. Microbiological testing showed the presence of known periodontal pathogens including Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Porphyromonas gingivalis. Routine immunological tests did not reveal any of the functional defects thought to play a role in the pathogenesis of EOP After initiation of therapy, which consisted of scaling and root planing, supplemented with administration of systemic antibiotics, a reduction in probing depth and gain in clinical attachment could be demonstrated. Microbiological testing was used to monitor the composition of the periodontal microbiota and to adjust antimicrobial therapy accordingly. RESULTS: Using a non-surgical approach to treatment, except for 2 root amputations performed without flap reflection, we have been able to stabilize this patient's periodontal condition over the course of a 2-year follow-up period. CONCLUSIONS: This treatment strategy provides an efficacious alternative to more aggressive forms of therapy and should therefore be considered for the treatment of patients with severe EOP.

Perturbation of mitochondrial structure and function plays a central role in Actinobacillus actinomycetemcomitans leukotoxin-induced apoptosis.

Certain pore-forming bacterial toxins, including the leukotoxin (Ltx) produced by Actinobacillus actinomycetemcomitans, induce apoptosis in susceptible target cells. Although binding to the target cell surface represents the first step in the initiation of this process, the downstream events leading to toxin-induced apoptotic cell death have not been identified. Perturbation of mitochondrial function has been shown to have a major role in regulating progression of apoptosis initiated by exposure to numerous stimuli. Using Ltx as a model, the aim of this study was to evaluate whether induction of apoptosis by pore-forming toxins follows a similar paradigm. After exposure to Ltx, Epstein-Barr virus transformed B cells (JY cell line) exhibited the classical morphological features of apoptosis including decreased cell size, plasma membrane blebbing, selective alterations in plasma membrane permeability and condensation of nuclear DNA. The morphologic changes were accompanied by swelling of the mitochondria, a decrease in mitochondrial transmembrane potential (Psi(m)), hyperproduction of reactive oxygen intermediates (ROIs) and release of cytochrome c from the intermembrane space. Subsequently, we detected activation of the c ysteine asp artate-specific prote ases (caspases)-3 and -9, cleavage of the nuclear DNA repair enzyme, poly(ADP-ribose)polymerase (PARP) and internucleosomal DNA fragmentation. These results indicate that perturbation of mitochondrial structure and function, in concert with activation of specific caspases, initiate the effector phase of Ltx-induced apoptosis.

Analysis of in situ protease activity in chronic adult periodontitis patients: expression of activated MMP-2 and a 40 kDa serine protease.

BACKGROUND: Periodontitis is characterized by extensive destruction of the gingival tissues and associated supporting structures of the teeth. Although the pathogenesis of the various forms of this disease is not completely understood, host-derived proteases are believed to have an important role. In this study, we analyzed human tissue samples from chronic adult periodontitis patients to assess the levels of specific proteases and determine the effect of pH and tetracyclines on their activity. METHODS: Gingival tissue samples were obtained from patients with chronic adult periodontitis (probing depths ranged from 5 to 9 mm) and periodontally healthy controls. Tissue extracts were prepared and analyzed for protease activity by zymography and Western blotting. RESULTS: Maximal protease activity from clinically normal and diseased tissues was observed at pH 8. Latent matrix metalloproteinase (MMP)-9 and MMP-2 were expressed in all samples examined, while active MMP-2 was detected only in tissues obtained from patients with clinical disease. The MMP activities were differentially inhibited by derivatives of tetracycline. At pH 6, a protease with a mass of approximately 40 kDa was observed in diseased samples. The enzymatic activity was inhibited by phenylmethylsulfonyl fluoride, suggesting it is a serine protease. CONCLUSIONS: The results of the current study substantiate the proposed role of host-derived proteases in the pathogenesis of chronic adult periodontitis. Specifically, they indicate that activated MMP-2 and a 40 kDa serine protease are involved in tissue destruction associated with this form of periodontal disease and also suggest that tissue pH influences protease activity in situ.

In situ localization and characterization of active proteases in chronically inflamed and healthy human gingival tissues.

BACKGROUND: Studies have indicated an important role for host-derived proteases in the pathogenesis of periodontal disease. The objectives of this study were: 1) to develop an assay measuring protease activity in situ and 2) to localize and characterize the enzymatic activity in intact inflamed and healthy gingiva. METHODS: Gingival specimens were prepared and over-laid with a quenched fluorescent substrate. Protease activity was visualized by fluorescence microscopy and correlated with histologic features. RESULTS: In inflamed tissues, enzymatic activity was detected mainly in the connective tissue (predominantly matrix metalloproteases) and, to some extent, in the epithelium (predominantly serine proteases). In contrast, clinically healthy tissues failed to exhibit significant amounts of protease activity. Quantitative and qualitative characteristics of protease activity in intact tissues were found to be pH dependent. CONCLUSIONS: The method described here enabled assessment of active proteases in intact tissues where cell-cell and cell-matrix interactions had been maintained. Our results indicate that there are substantial differences in the distribution of specific proteases between clinically healthy and inflamed periodontal tissues.

The interaction between RTX toxins and target cells.

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They fall into two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins, which are cell-type- and species-specific. These toxins offer interesting models for targeting, insertion and translocation of aqueous proteins into lipid membranes.

Actinobacillus actinomycetemcomitans leukotoxin induces apoptosis in HL-60 cells.

Actinobacillus actinomycetemcomitans leukotoxin (Ltx) is a member of the repeats-in-toxin (RTX) family of pore-forming toxins and kills human immune cells. Currently, it remains unclear whether toxin-mediated killing of target cells involves the induction of necrosis or apoptosis. Therefore, the goal of this investigation was to determine whether Ltx is capable of causing apoptotic cell death in toxin-sensitive promyelocytic HL-60 cells. Multiparameter flow cytometric analysis of toxin-treated cells stained with Hoechst 33258 (or 33342) and 7-aminoactinomycin D allowed us to identify four populations: viable cells, early apoptotic cells, late apoptotic and/or secondarily necrotic cells, and a final population that was composed of cellular debris. Compared with control cells, HL-60 cells treated with Ltx exhibited a gradual decrease in forward light scatter with a coincident increase in side light scatter, indicative of a decrease in cell size and organelle condensation, respectively. Additional experiments demonstrated that Ltx-treated cells showed evidence of internucleosomal DNA fragmentation and phosphatidylserine translocation. The results of our studies clearly demonstrate that Ltx can kill HL-60 cells by inducing apoptosis. We hypothesize that elimination of acute inflammatory cells via this mechanism plays a critical role in the pathogenesis of diseases caused by A. actinomycetemeomitans.

Mechanical forces alter extracellular matrix synthesis by human periodontal ligament fibroblasts.

Periodontal ligament fibroblasts (PDLFs) are a heterogeneous population of cells that are involved in the normal maintenance, repair and regeneration of both the ligament and adjacent hard tissues. Additionally, the ability of these cells to respond to mechanical stimulation suggests that they have a central role in mediating the osseous remodeling that underlies physiological and orthodontic tooth movement. To characterize their role further in this process, the current study evaluated the effect of tensional stress on the biosynthesis of extracellular matrix (ECM) proteins by human PDLFs. Cell strains were established from extracted human premolars and third molars. Cells exposed to 5% biaxial deformation (strain) at a frequency of 30 times/min for 24 hr exhibited statistically significant increases in type I collagen and fibronectin synthesis, and a statistically significant decrease in tropoelastin production relative to unstretched controls. Cells exposed to 10% strain exhibited similar responses for fibronectin and tropoelastin while the amount of type I collagen synthesized by stretched cells did not differ from control levels. These results indicate that mechanical stimulation of PDLFs alters type I collagen, tropoelastin and fibronectin production and that these cells are differentially responsive to varying levels of mechanical stress. The ability of these cells to alter ECM protein synthesis in response to specific magnitudes of tensional stress may in part explain how PDLFs regulate ligament and hard tissue remodeling.

Molecular and biochemical mechanisms of Pasteurella haemolytica leukotoxin-induced cell death.

Pasteurella haemolytica leukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a beta2 integrin since pre-incubating cells with anti-beta2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.

RTX toxins recognize a beta2 integrin on the surface of human target cells.

Actinobacillus actinomycetemcomitans leukotoxin and Escherichia coli alpha-hemolysin are RTX toxins that kill human immune cells. We have obtained a monoclonal antibody (295) to a cell surface molecule present on toxin-sensitive HL60 cells that can inhibit cytolysis by both RTX toxins. Utilization of this monoclonal antibody for immunoaffinity purification of detergent-solubilized target cell membranes yielded two polypeptide chains of approximate molecular masses of 100 and 170 kDa. Microsequencing of tryptic peptides from the two proteins showed complete homology with CD11a and CD18, the two subunits of the beta2 integrin, lymphocyte function-associated antigen 1 (LFA-1). Anti-CD11a and CD18 monoclonal antibodies also inhibited RTX toxin-mediated cytolysis. Direct binding experiments demonstrated the ability of an immobilized RTX to bind LFA-1 heterodimers present in a detergent lysate of human HL60 target cells. Transfection of CD11a and CD18 integrin genes into a cell line (K562) that is not sensitive to either RTX toxin resulted in LFA-1 expressing cells, KL/4, that were sensitive to both toxins. These experiments identify LFA-1 as a cell surface receptor that mediates toxicity of members of this family of pore-forming toxins.

Manipulation of the immune response by foreign gene expression in the thymus.

Retroviral gene transfer vectors have been developed for optimal in vivo gene therapy. Ideally, these vectors should target gene expression specifically to selected tissues or organs. Our studies focus on the development of retroviral vectors for gene delivery to the thymus. The goal of these studies is to utilize thymic expression of exogenous genes to manipulate the immune repertoire. We have characterized the selective thymic tropism of a molecular clone of Gross murine leukemia virus, GD-17, to thymic medullary epithelial cells using immunohistochemical staining and confocal microscopy. Specific expression of viral antigens in the thymus lead to the induction of immunologic tolerance to GMuLV proteins. This tissue specific vector may thus be used to study the requirements of epithelial mediated tolerance induction, and provide a more efficient tool for gene therapy.

Impaired immune responsiveness is an essential component in persistent central nervous system infection with gross murine leukemia virus.

Exposure of newborn mice to Gross murine leukemia virus (GMuLV) results in persistent viral infection of the central nervous system (CNS) white matter. Animals exposed to virus as neonates showed a marked depression in GMuLV-specific B lymphocyte function as evidenced by significant decreases in adult and neonatal anti-GMuLV antibody levels. Immunohistochemical analyses showed that the sites of GMuLV infection in the CNS were also devoid of major histocompatibility complex (MHC) class I and II protein expression, although transplantation of GMuLV-infected brain tissue to the kidney capsules of immunocompetent mice induced a potent mononuclear cell graft infiltrate. These results indicate that persistent GMuLV infection of the CNS is linked to both impairment of anti-GMuLV peripheral immune responses and deficient antigen-presenting cell function within the CNS.