Antibody-Mediated Osseous Regeneration for Bone Tissue Engineering in Canine Segmental Defects.

Among many applications of therapeutic monoclonal antibodies (mAbs), a unique approach for regenerative medicine has entailed antibody-mediated osseous regeneration (AMOR). In an effort to identify a clinically relevant model of craniofacial defect, the present study investigated the efficacy of mAb specific for bone morphogenetic protein- (BMP-) 2 to repair canine segmental mandibular continuity defect model. Accordingly, a 15 mm unilateral segmental defect was created in mandible and fixated with a titanium plate. Anorganic bovine bone mineral with 10% collagen (ABBM-C) was functionalized with 25 µg/mL of either chimeric anti-BMP-2 mAb or isotype-matched mAb (negative control). Recombinant human (rh) BMP-2 served as positive control. Morphometric analyses were performed on computed tomography (CT) and histologic images. Bone densities within healed defect sites at 12 weeks after surgery were 1360.81 ± 10.52 Hounsfield Unit (HU), 1044.27 ± 141.16 HU, and 839.45 ± 179.41 HU, in sites with implanted anti-BMP-2 mAb, rhBMP-2, and isotype mAb groups, respectively. Osteoid bone formation in anti-BMP-2 mAb (42.99% ± 8.67) and rhBMP-2 (48.97% ± 2.96) groups was not significantly different but was higher (p < 0.05) than in sites with isotype control mAb (26.8% ± 5.35). In view of the long-term objective of translational application of AMOR in humans, the results of the present study demonstrated the feasibility of AMOR in a large clinically relevant animal model.

The in vivo T helper type 17 and regulatory T cell immune responses to Aggregatibacter actinomycetemcomitans.

The periodontal pathogen Aggregatibacter actinomycetemcomitans is known to elicit a systemic immune response in the infected host, and occasionally causes non-oral infections. Detailed information on its immunopathological responses and the involvement of bacterial virulence factors remains to be elucidated. The aim of this study was to assess the systemic immune response to A. actinomycetemcomitans oral infection. We used an animal model that simulates systemic dissemination of the bacteria by injecting live wild-type (WT) D7S-1 and a double knockout mutant of leukotoxin and cytolethal distending toxin (ΔltxΔcdt) A. actinomycetemcomitans strains in rat oral mucosa. Draining lymph nodes were examined for regulatory T (Treg) and T helper type 17 (Th17) cell subsets and their associated mediators. An increase in the proportion of Th17 cells and a decrease in Treg cells over the experimental period of 3 weeks were similarly observed for rats challenged with WT and ΔltxΔcdt. Significant upregulation and downregulation of proinflammatory cytokines in the Th17 gene pathway was noted, as well as several qualitative differences between WT and ΔltxΔcdt. Furthermore, we observed differential fold regulation in key genes associated with a proinflammatory response in ΔltxΔcdt-inoculated rats relative to D7S-1 group. This suggests that although the knockout of these two virulence factors (ΔltxΔcdt) may suppress certain proinflammatory genes, it causes similar over-expression of other genes compared with D7S-1, indicating a common factor that still remains in the pathogenicity of A. actinomycetemcomitans.

Novel calcified gum Arabic porous nano-composite scaffold for bone tissue regeneration.

The aim of this study was to investigate the biomechanical and biological properties of a nanocomposite scaffold containing both mineral and polysaccharide constituents. Hydroxyapatite nanoparticles (n-HA) was synthesized from dead abra ovata shells using wet chemical methods and was used in different ratios in concert with gum Arabic, a branched plant polysaccharide. N-HA/gum nanocomposite was fabricated with freeze-drying process and characterized by FTIR and SEM for chemical structure and morphology. Porosity was estimated using liquid substitution method. The scaffold mechanical properties were evaluated by compressive test measurement. Osteogenic differentiation was assessed using alkaline phosphatase production and biomineralization was evaluated using Alizarin red assay. Results demonstrated that the hydroxyapatite/gum Arabic nanocomposite had favorable biocompatibility and a similar structure to natural bone matrix. Porous nanocomposite possessed macropore networks with a porosity 87-93% and mean pore size ranging between 164 and 230 µm. The gum/HA with a ratio of 50% w/w HA had the highest compressive modulus of ∼75.3 MPa Pa (MPa) and the ultimate compressive stress of ∼16.6 MPa. C2C12 cells cultured on a scaffold with higher percentage (40 and 50 w/w) of HA demonstrated increased ALP levels and calcium deposition. The data from the present study demonstrated significant changes to the biomechanical properties and osteoconductivity of the nanocomposite scaffold by modulating its mineral content. Nanocomposite scaffolds containing gum and n-HA of 40-50% exhibited highest mechanical properties, as well as supported increased biomineralization.

A bacterial-biofilm-induced oral osteolytic infection can be successfully treated by immuno-targeting an extracellular nucleoid-associated protein.

Periodontal disease exemplifies a chronic and recurrent infection with a necessary biofilm component. Mucosal inflammation is a hallmark response of the host seen in chronic diseases, such as colitis, gingivitis, and periodontitis (and the related disorder peri-implantitis). We have taken advantage of our recently developed rat model of human peri-implantitis that recapitulates osteolysis, the requirement of biofilm formation, and the perpetuation of the bona fide disease state, to test a new therapeutic modality with two novel components. First we used hyperimmune antiserum directed against the DNABII family of proteins, now known to be a critical component of the extracellular matrix of bacterial biofilms. Second we delivered the antiserum as cargo in biodegradable microspheres to the site of the biofilm infection. We demonstrated that delivery of a single dose of anti-DNABII in poly(lactic-co-glycolic acid) (PLGA) microspheres induced significant resolution of experimental peri-implantitis, including marked reduction of inflammation. These data support the continued development of a DNABII protein-targeted therapeutic for peri-implantitis and other chronic inflammatory pathologies of the oral cavity in animals and humans.

Perturbation of the indigenous rat oral microbiome by ciprofloxacin dosing.

Mucosal surfaces such as the gut, vagina and oral cavity are colonized by microbiota that are an integral component of the healthy ecosystem. Recent molecular techniques make it feasible to correlate antimicrobial dosing levels with changes in microbiome composition. The objective of this study was to characterize the rat oral plaque microbiome composition at doses of ciprofloxacin that were considerably above and below nominal in vitro minimal inhibitory concentrations of a variety of gram-positive oral commensal bacteria. We exposed the oral cavities of rats to relatively low (0.1 µg ml(-1) ) and high (20 µg ml(-1)) doses of ciprofloxacin in the drinking water over a 3-day period. Plaque microbiota were characterized using 454 pyrosequencing. The rat indigenous community was dominated by the genera Rothia (74.4%) and Streptococcus (4.7%). Dosing at 0.1 µg ml(-1) was associated with changes in Rothia and Streptococcus species that were not significant, whereas dosing at 20 µg ml(-1) caused a pronounced (significant) reduction in the relative abundance of the Streptococcus genus. Taxonomic independent analysis indicated that the perturbation in the overall community structure attributed to dosing with ciprofloxacin at either the low or high dose was relatively low. The results suggest that it is feasible to use an antimicrobial dosing regimen to selectively target a specific subset of a mucosal microbiome for elimination with minimal perturbation of the entire community.

Induction of T-cell apoptosis by Actinobacillus actinomycetemcomitans mutants with deletion of ltxA and cdtABC genes: possible activity of GroEL-like molecule.

The pathogenic bacterium Actinobacillus actinomycetemcomitans expresses a leukotoxin (Ltx) and cytolethal distending toxin (CDT) with cytolytic properties. CDT also has cytostatic properties, inducing a G2 cell cycle block. The extent of the contribution of these, as well as other toxins, to the cytolytic and cytostatic activities of this microorganism have not been defined and the aim of this study was to determine their contribution. To that end, a naturally transformable A. actinomycetemcomitans clinical strain (D7S-smooth) was used to construct a series of deletion mutants (DeltacdtA, DeltacdtB, DeltacdtC, DeltacdtABC, DeltaltxA, DeltaltxA/DeltacdtABC). Human peripheral blood mononuclear cells were incubated with cell-associated and extracellular bacterial preparations. The ability of wild type and isogenic mutants to induce T-cell apoptosis and cell cycle arrest was compared. The expression of ltxA and each of the cdt gene loci partially contributed to A. actinomycetemcomitans apoptosis, since each of the isogenic mutants exhibited reduced ability to induce T-cell apoptosis. Conversely, the ability to induce cell cycle block was abolished in each of the cdt isogenic mutants. A mutant with simultaneous deletion of ltxA and cdtABC genes retained potent ability to induce apoptosis in its cell-associated, but not extracellular, preparation. Neutralization with Escherichia coli anti-GroEL monoclonal antibody, lead to significant diminution of apoptosis-inducing activity of the DeltaltxA/DeltacdtABC cell-associated preparation. These data provide evidence for the expression of other A. actinomycetemcomitans cytolytic molecule(s) distinct from CDT and leukotoxin, with a possible role for GroEL-like molecule in T-cell apoptosis.

Actinobacillus actinomycetemcomitans induces apoptosis of T lymphocytes by the Fas and Fas ligand pathway.

Actinobacillus actinomycetemcomitans expresses a number of toxins capable of inducing apoptotic cell death of T lymphocytes. However, the exact mechanism(s) has not been elucidated. The present study investigated the involvement of the Fas (CD95)-mediated apoptotic pathway in A. actinomycetemcomitans-induced T-cell apoptosis. To that end, peripheral blood mononuclear cells (PBMC) were cultured with or without A. actinomycetemcomitans cell-free culture supernatant (CFCS) for 0-96 h. The cells were then labeled with specific monoclonal antibodies and flow cytometry was performed. Results demonstrated up-regulation of Fas and activation of caspase-3 in T cells in response to A. actinomycetemcomitans CFCS. Monocytes were the only cells analyzed to express Fas ligand (FasL) constitutively, and this was further up-regulated in response to A. actinomycetemcomitans CFCS, while T cells expressed FasL only after this stimulation. Depletion of monocytes prior to stimulation with A. actinomycetemcomitans CFCS led to a marked decline in apoptosis. Blocking of Fas-FasL interactions with anti-Fas monoclonal antibody or Fas:Fc fusion protein lead to a significant decline, but not abolition, of T-cell apoptosis. Nearly all T cells expressed Bcl-2 at the outset of culture, and Bcl-2 expression declined in T cells stimulated with A. actinomycetemcomitans CFCS. Collectively, these data provide evidence for the induction of T-cell apoptosis by A. actinomycetemcomitans via the Fas-mediated pathway, involving caspase-3 and Bcl-2. Moreover, this apoptotic response was dependent on the presence of monocytes.

The effect of periodontal treatment on glycemic control in patients with type 2 diabetes mellitus.

BACKGROUND, AIMS: This study was designed to explore the effect of periodontal therapy on glycemic control in persons with type 2 diabetes mellitus (DM). METHODS: 36 patients with type 2 DM (treatment group) received therapy for adult periodontitis during an 18-month period. A 36-person control group was randomly selected from the same population of persons with type 2 DM who did not receive periodontal treatment. RESULTS: These groups were well matched for most of the parameters investigated. During the nine-month observation period, there was a 6.7% improvement in glycemic control in the control group when compared to a 17.1% improvement in the treatment group, a statistically significant difference. Several parameters that could confound or moderate this glycemic control were explored. These included the treatment of non-dental infections, weight and medication changes. No moderating effect was associated with any of these variables. However, there were too few subjects in the study to have the statistical power necessary to assess these possible moderators of glycemic control. CONCLUSIONS: We interpret the data in the study to suggest that periodontal therapy was associated with improved glycemic control in persons with type 2 DM.

Large-scale early in vitro response to actinobacillus actinomycetemcomitans suggests superantigenic activation of T-cells.

The mode of T-cell response to Actinobacillus actinomycetemcomitans is largely unknown. The present study sought to investigate the hypothesis that A. actinomycetemcomitans expresses superantigens, capable of antigen-non-specific T-cell activation. To that end, peripheral blood mononuclear cells were stimulated with A. actinomycetemcomitans, and T-cell expression of the early activation marker, CD69, was determined by flow cytometry. Results showed that A. actinomycetemcomitans activated a large number of T-cells with magnitude similar to that of staphylococcal enterotoxin superantigens. A. actinomycetemcomitans sonicate preferentially activated T-cells expressing Vbeta5.1 and Vbeta8, while the extracellular preparation activated Vbeta5.1+, Vbeta8+, and Vbeta12+ T-cells. T-cell response to A. actinomycetemcomitans was observed in the presence of autologous, as well as heterologous, antigen-presenting cells, suggesting a MHC-non-restricted response. Thus, the in vitro response to A. actinomycetemcomitans is characterized by large-scale T-cell activation in a Vbeta-specific and MHC-non-restricted manner, consistent with the involvement of superantigens.

Despite large-scale T cell activation, only a minor subset of T cells responding in vitro to Actinobacillus actinomycetemcomitans differentiate into effector T cells.

Recent studies in our laboratory have demonstrated that Actinobacillus actinomycetemcomitans has a potent T cell stimulatory effect, activating more than half of all T cells. However, since the fate of these activated T cells was not known, the present study sought to determine whether all of these T cells differentiate into effector cells. To that end, the intracellular expression of T cell cytokines (IL-2, IFN-gamma, IL-4 and IL-10) in response to A. actinomycetemcomitans was determined by flow cytometry. Results demonstrated a time-dependent increase in the expression of the cytokines, most reaching peak levels at 24-48 h. At 48 h, the proportion of T cells expressing each of the cytokines were as follows: IL-2 (1.7%+/-0.3), IFN-gamma (1.8%+/-0.5), IL-4 (1.0%+/-0.2) and IL-10 (1.5%+/-0.5). These data indicated that only 2-5% of all T cells stimulated with A. actinomycetemcomitans expressed any T cell cytokines. The finding of large-scale T cell activation in the absence of cytokine expression suggests that the activation of T cells in response to A. actinomycetemcomitans is incomplete. To investigate this phenomenon, peripheral blood mononuclear cells (PBMC) were cultured with A. actinomycetemcomitans for 24 h followed by sorting of the activated (CD69+) cells by immunomagnetic separation and restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results demonstrated that nearly 90% of the T cells were unresponsive to further restimulation. A possible explanation for this unresponsiveness is the induction of clonal anergy among the responding T cells. To determine possible preferential effects of the stimulation on specific cytokines, the expression of each cytokine among T cells responding to A. actinomycetemcomitans was compared to the maximum levels achieved by PMA + ionomycin stimulation. Results showed that number of IL-2+ and IFN-gamma+ T cells observed in response to A. actinomycetemcomitans were between 2% and 7% of those seen in response to PMA + ionomycin. Conversely, the proportions of T cells expressing IL-4 or IL-10 were between 35% and 90% of those following stimulation with PMA + ionomycin. Hence, A. actinomycetemcomitans appears to more preferentially induce T cells expressing IL-4 and IL-10. Collectively, these data suggest that the in vitro stimulation of T cells with A. actinomycetemcomitans leads to partial activation, i.e. only a minor subset of T cells responding to A. actinomycetemcomitans differentiate into effector cells, while a significant proportion become unresponsive to restimulation, suggesting clonal anergy.

Evidence for apoptosis of the majority of T cells activated in vitro with Actinobacillus actinomycetemcomitans.

Our previous studies had demonstrated that nearly half of all T cells stimulated with Actinobacillus actinomycetemcomitans are activated within a few hours. However, it was not known whether all of these T cells survive. The aim of the present study was to determine whether the T cells activated in response to A. actinomycetemcomitans undergo apoptosis. To that end, peripheral blood mononuclear cells were cultured at different time points in the presence of A. actinomycetemcomitans. Flow cytometric analysis demonstrated that, following exposure to a preparation of A. actinomycetemcomitans, T cells progressively externalized their plasma membrane phosphatidylserine, as measured by annexin V binding. Approximately half of all T cells bound annexin V by 96 h. During this period, Annexin V-positive T cells also incorporated propidium iodide suggesting loss of membrane integrity. The externalization of phosphatidylserine occurred at a higher rate among activated (CD69+) T cells, where roughly two-thirds became Annexin V-positive. Flow cytometric analysis also demonstrated shrinkage of the Annexin V-positive and propidium iodide-positive T cells. The data presented here provides evidence for the induction of apoptosis among the majority of the T cells responding to A. actinomycetemcomitans.

Potential adjunctive applications of hypnosis in the management of periodontal diseases.

Many uses of hypnosis in dentistry have been described in the literature including anesthesia, analgesia, anxiety management, treatment for bruxism, to control gagging, and the alteration of salivary flow and bleeding control during treatment. However, very few references have been made specifically regarding the use of hypnosis with patients who have periodontal disease, a wide spread chronic inflammatory disease affecting the oral cavity of about 80% of the population. The purpose of this paper is to describe potential adjunctive applications of hypnosis in the treatment of patients with periodontal diseases. The supporting literature from two broad areas for potential application, health behaviors and psychoneuroimmunology is discussed, followed by proposed hypnotic strategies and suggestions for use with patients with periodontal diseases.

Herpesvirus infection of inflammatory cells in human periodontitis.

Human cytomegalovirus (HCMV) and Epstein-Barr virus type 1 (EBV-1) are frequently detected in human periodontitis lesions. However, no information is available on the types of gingival cells infected by herpesviruses. The present study determined the presence of herpesviruses in polymorphonuclear neutrophils, monocytes, macrophages and T and B lymphocytes in biopsies of periodontitis lesions from 20 adults. A nested polymerase chain reaction method was employed to detect HCMV, EBV-1, EBV-2, human herpes virus-6 and herpes simplex virus (HSV) in periodontal tissue biopsy and in gingival cell fractions separated by immunomagnetic cell sorting. Tissue specimens from 18 (90%) and cell fractions from 14 (70%) patients demonstrated herpesviruses. Periodontitis-derived monocytes and macrophages revealed HCMV in cell fractions from 11 (55%) patients and HSV in cells from 1 (5%) patient. T lymphocytes harbored HCMV in cell fractions from 4 (20%) patients and HSV in cell fractions from 4 (20%) patients. B lymphocytes showed EBV-1 in cell fractions from 9 (45%) patients. Periodontal polymorphonuclear neutrophils demonstrated no herpesviruses. This study suggests that HCMV mainly infects periodontal monocytes, macrophages and less frequently T lymphocytes and that EBV-1 infects periodontal B lymphocytes. The possible etio-pathologic significance of periodontal herpesvirus infection is discussed.

Comparison of gingival and peripheral blood T cells among patients with periodontitis suggests skewing of the gingival T cell antigen receptor V beta repertoire.

The present study investigated the expression of different variable regions of T cell receptor beta-chain (V beta) among functional subsets of T cells, i.e. CD45RO+ (activated/memory), CD4+ and CD8+ in gingiva and peripheral blood of patients with periodontitis. Gingival tissue specimens (n = 25) and peripheral blood were procured from 18 patients with periodontitis during periodontal surgery or extraction. Single-cell suspensions of gingival tissues were made by enzymatic digestion. These cells were immunofluorescently labeled with a panel of monoclonal antibodies specific for 18 TCR V beta regions, in concert with markers for various T cell subsets. The cells were then analyzed with 3-color multivariate flow cytometry. Results demonstrated that a significantly higher proportion of T cells in gingiva expressed V beta 5.2 (0.0005), V beta 6 (0.0007) and V beta 9 (0.003) regions compared to those in peripheral blood. Comparison of CD45RO+ (activated/memory) and CD45RO- (naïve) subsets of gingival T cells revealed differences in the expression of TCR V beta regions. V beta 5.2 expression was significantly higher among CD45RO+ gingival T cells (p = 0.004), whereas V beta 14 expression was elevated among the CD45RO- subset relative to peripheral blood (p = 0.008). Analysis of TCR V beta region expression among CD4+ and CD8+ subsets did not reveal any statistically significant differences between gingiva and peripheral blood, although some V beta regions approached significance. Collectively, these results demonstrate that the T cell repertoire in the gingival compartment differs significantly from that in the peripheral blood. Furthermore, since the skewing of TCR V beta was observed among naïve, as well as activated/memory T cells, it is likely that both developmental and environmental factors are influential in shaping the gingival TCR repertoire in patients with periodontitis. Elucidation of the cause of the skewed expression of T cell receptors in gingiva can provide insights into the specificity of T cells in periodontitis.

Evidence for involvement of superantigens in human periodontal diseases: skewed expression of T cell receptor variable regions by gingival T cells.

Immunomodulation by periodontopathic bacteria has been implicated in the pathogenesis of inflammatory periodontal diseases. A novel class of microbial-derived T cell mitogens, referred to as superantigens, has recently been described. Superantigens are unique in that they induce a tremendous activation and expansion of specific subsets of T cells in an antigen-independent manner, thereby causing immune dysfunction. Subsets of superantigen-expanded T cells can be identified with reagents that discriminate among different families of the variable domains of the T cell antigen receptor beta-chain (V beta). Since superantigens expand one or a few of these T cell antigen receptor V beta families, T cell subsets that have been expanded by superantigens have restricted expression of one or a few V beta families. In the present study, we investigated the presence of putative superantigen-stimulated T cells in periodontitis sites, utilizing a panel monoclonal antibodies to T cell antigen receptor V beta families. Leukocytes were isolated from gingival tissues obtained from 8 periodontitis and 4 non-periodontitis patients by collagenase digestion. Three-color flow cytometric analysis of these gingival cells demonstrated that in most periodontitis patients examined, patterns of V beta expression among T cells are characteristic of superantigen stimulation, i.e., there is an elevation in the proportion of one or a few V beta families. Specifically, these analyses revealed that T cell subsets expressing V beta 5a and V beta 5b, V beta 6, V beta 8 and V beta 12 were each elevated greater than 2 standard deviations in at least one periodontitis patient compared with the mean of the non-periodontitis subjects. In some periodontitis patients, a less marked elevation of T cells that express V beta 3, V beta 5a, V beta 5b, V beta 6, V beta 8, V beta 12, and V beta 13 was noted (greater than 1 standard deviation higher than the mean of the V beta families in non-periodontics subjects). Interestingly, V beta 8+ T cells were elevated to some degree in all periodontitis patients examined. In contrast, T cells expressing V beta 2, V beta 17 and V beta 19 were not significantly different in any of the subjects studied. In most periodontitis but not non-periodontitis patients, up to 50% of all gingival T cells expressed one or a few T cell antigen receptor V beta families, suggesting that superantigens constitute a major pathway of T cell activation and expansion. Hence, our data support the hypothesis that a large proportion of T cells in periodontitis sites have been stimulated and expanded by superantigens, presumably produced by periodontitis-associated bacteria.

Abnormalities in the export and fate of recent thymic emigrants in diabetes-prone BB/W rats.

Abnormalities in postthymic T cell development in the BB/W rat model of autoimmune insulin-dependent diabetes mellitus (IDDM) result in part from a lymphopenia (lyp) gene defect. To better characterize these abnormalities, the phenotypes of T cells from diabetes-prone (DP) and diabetes-resistant (DR) coisogenic rats were analyzed by multiparameter flow immunocytometry (FCM). Marked decreases in the numbers of Thy1- RT6+ T cells, most of which are CD8+, were documented in DP rats by live-gating. Conversely, an approximately 3-fold increase was observed in the percentage of Thy1+ RT6- T cells, which normally serve as the precursors of both Thy1- RT6+ and Thy1- RT6- T cell subsets in rats. These results suggested that, at a minimum, an arrest in maturation of the Thy1+ precursors of RT6+ T cells occurs postthymically in DP rats. To determine more precisely the stage(s) in T cell development at which lymphopenia occurs, the export and fate of recent thymic emigrants (RTE's) and their immediate descendants in DP rats was traced after intrathymic (i.t.) labelling with fluorescein isothiocyanate (FITC). The results showed that in DP, as compared with DR, rats: 1) 5-fold fewer RTE's are exported from the thymus per 24 hr; 2) more than 80% of the RTE's are CD4+; 3) most of the immediate descendants of RTE's disappear from the peripheral lymphoid tissues within one week after export from the thymus; and 4) few of the descendants of the RTE's that do survive differentiate into RT6+ T cells. Staining with propidium iodide revealed that a significantly higher proportion of Thy1+ T cells in DP than in DR rats are in cycle (S/G2/M), thereby accounting for their disproportionately high numbers relative to RTE's. These results indicate that, in addition to defective thymic export, most of the immediate descendants of RTE's in DP rats undergo non-productive proliferation and death at the time (3-7 days postthymic) at which their counterparts in DR rats differentiate into Thy1- RT6+ T cells. The resulting deficiency of immunoregulatory T cells, acting in concert with defective intrathymic selection of effector T cell precursors, appears to conspire to markedly enhance the predisposition of DP rats to autoimmunity.

Demonstration of large-scale migration of cortical thymocytes to peripheral lymphoid tissues in cyclosporin A-treated rats.

Young adult Lewis rats were maintained on diets containing 0.015 or 0.027% cyclosporin A (CSA) for periods of up to 6 wk. All animals showed complete depletion of medullary thymocytes (CD4+8- and CD4-8+, T cell receptor [TCR] alpha/beta hi, Thy-1med/low, terminal deoxynucleotidyl transferase negative [TdT-]) and a 50% reduction in the number of TdT- cortical thymocytes (CD4+8+, TCR alpha/beta low, Thy-1med) within 1 wk of CSA treatment. In addition, about half of the animals displayed a 50% reduction in the number of TdT+ cortical thymocytes (CD4+8+, TCR alpha/beta low, Thy-1hi). These intrathymic changes were accompanied by a reciprocal increase in the number of double-positive (DP; CD4+8+) T cells in lymph nodes (LN) and spleens. To confirm that the latter T cells were recent thymic emigrants (RTE), CSA-treated rats were injected intrathymically with fluorescein isothiocyanate, and the phenotype of the labeled T cells appearing in LN was determined 16 h later. The results demonstrated that, in addition to those RTE exported in normal animals (> 90% medullary origin), the emigration of DP thymocytes, including large numbers of TdT+ thymocytes, was markedly increased. The presence of TdT+ cells, which normally do not leave the thymus, clearly identifies the DP RTE as originating from the thymus cortex. Intrathymic labeling studies also directly demonstrated that export of all thymocyte subsets ceases within 9 d of CSA treatment; and thymectomy experiments confirmed that the CSA-induced increase in phenotypically immature T cells resulted primarily from the disturbance of thymocyte maturation and emigration, rather than from a direct effect on preexisting T cells. These results suggest that a wave of cortical thymocytes, many of which presumably have not yet undergone negative selection, is released from the thymus during the first week of CSA treatment. The presence of these potentially unselected cells in peripheral lymphoid tissues may help to explain the increased frequency of autoreactive T cells observed in CSA-treated animals.