Interaction of oral bacteria with gingival epithelial cell multilayers.

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low-calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1ß and TNF-α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.

Rituximab inhibits structural joint damage in patients with rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitor therapies.

OBJECTIVE: To determine if treatment with a B cell-targeted therapy can inhibit the progression of structural joint damage in patients with rheumatoid arthritis (RA), exhibiting an inadequate response to tumour necrosis factor (TNF) inhibitors. METHODS: In this phase III study, patients with an inadequate response to a TNF inhibitor and receiving methotrexate were randomised to rituximab or placebo. Radiographs were obtained at baseline, week 24 and week 56 after randomisation. Patients with an inadequate response to their randomised therapy could receive rescue medication from week 16. From week 24, eligible patients from both treatment arms could receive open-label rituximab. Patients were analysed according to their original treatment group. Radiographs were scored using the Genant-modified Sharp method. The primary radiographic endpoint was change in total Genant-modified Sharp score at week 56. RESULTS: Rituximab treatment caused significant reduction in joint damage progression compared with placebo. The mean change from baseline in the total Genant-modified Sharp score at week 56 was significantly lower for patients treated with rituximab than for patients treated with placebo (1.00 vs 2.31; p = 0.005), and was supported by changes in erosion score (0.59 and 1.32 for rituximab plus methotrexate vs placebo plus methotrexate, respectively; p = 0.011) and joint space narrowing score (0.41 and 0.99, respectively; p<0.001). CONCLUSIONS: This study provides the first evidence that a B cell-targeted therapy-rituximab-can significantly inhibit the progression of structural joint damage in patients with RA with long-standing, active and treatment-resistant disease.

Very high and increasing incidence of type 1 diabetes mellitus in Newfoundland and Labrador, Canada.

OBJECTIVE: To determine the incidence of type 1 diabetes mellitus (T1DM) among children aged 0-14 yr inclusive in the Canadian province of Newfoundland and Labrador (NL). METHODS: Prospective and retrospective cohort study of the incidence of T1DM in children aged 0-14 yr from 1987 to 2005. Identified cases during this time period were ascertained from several sources and verified using the capture-recapture technique. RESULTS: Over the study period, 732 children aged 0-14 yr were diagnosed with T1DM. The incidence of T1DM in this population over the period 1987-2005 inclusive was 35.08 per 100,000 (95% confidence interval: 32.54, 37.62). The incidence over this period increased linearly at the rate of 0.78 per 100 000 per year. There was a significant difference between the incidence of 31.61 per 100,000 for boys in the 0-4-yr age-group and 19.05 per 100,000 for girls in the 0-4-yr age-group (p = 0.001). The incidence was very high throughout the entire province. CONCLUSIONS/INTERPRETATION: The province of NL has one of the highest incidences of T1DM reported worldwide. The incidence is increasing over the 19-yr study period.

A signalling accessory molecule revealed by a new anti-fibroblastoid L cell monoclonal antibody.

Fibroblastoid mouse L-cells are widely used in immunological models because when transfected with class II-coding genes they become efficient antigen presenting cells. Little is known, however, about the cell surface markers borne by L-cells and their putative involvement/Interference with the experimental models studied. Rats were immunized against DAP.3 cells (subclone of L-cells) and monoclonal antibodies (mAbs) were prepared. One of them, 4D4, was studied in detail. It recognizes an epitope which is neither cell lineage- nor strain- nor species-restricted since, in addition to DAP.3 cells, it binds, as determined by flow cytometry and immunohistochemistry, to various cells such as CD8+ T cells from thymus, spleen, lymph node or intestinal epithelium, mouse peritoneal B cells and various tissues such as renal, pulmonary or intestinal epithelia. 4D4 mAb immunoprecipitates an undescribed 68 kDa protein. Functionally, this mAb inhibits the IL-2 secretion of a T cell clone in response to its peptide presented by appropriate class II-transfected L-cells and induces a negative selection of double positive CD4+CD8+ thymocytes. Since the 4D4 ligand is found on cells which are submitted to selection (T cells) and on cells which mediate selection (epithelial and antigen presenting cells), we conclude that 4D4 mAb defines a cell surface antigen involved, as an accessory molecule, in a cell selection process.

Intercellular adhesion molecule-1 is necessary but not sufficient to activate CD4+ T cells. Discovery of a novel costimulator on kidney tubule cells.

Kidney tubule cells (KTC) are targets of T lymphocyte injury during allograft rejection and interstitial nephritis. KTC process and present self- and foreign Ags for immune recognition by CD4+ T cells in vivo and in vitro. However, it is not known whether KTC can provide the costimulatory signal required to fully activate CD4+ T cells. Using the MRL/MpJ fas model of lupus interstitial nephritis, we found that KTC did not express the costimulators B7-1 or B7-2. Nevertheless, KTC from both normal and systemically infected mice provided non-B7 costimulation to splenic CD4+ T cells. T cell proliferation was blocked by mAbs binding intercellular adhesion molecule-1 (ICAM-1) but not by mAb or fusion proteins binding B7-1, B7-2, heat-stable Ag, or vascular cell adhesion molecule-1. Importantly, ICAM-1 expression was necessary but not sufficient to provide costimulation. The transformed KTC line D3.B7- expressed high levels of ICAM-1 but did not provide costimulation. Interestingly, KTC provided costimulation to splenic T cells but not to a Th1 clone. These results show that freshly isolated KTC can provide non-B7 costimulation to splenic T cells via an unidentified costimulator and ICAM-1. Furthermore, these experiments demonstrate the complex nature of T cell activation and show that at least for splenic T cells, three or more signals may be required for full activation on live APC.

Living-unrelated renal transplantation provides comparable results to living-related renal transplantation: a 12-year single-center experience.

BACKGROUND: The increasing success of renal transplantation is paralleled by the increased size of the waiting list. Efforts to increase the donor pool have included the use of living-unrelated kidney donors (LURDs). METHODS: During a 12-year period our center performed 309 transplantation from living donors; 279 patients received living-related donor (LRD) transplants, and 30 patients received LURD transplants. During the same period 543 patients received cadaveric renal donor transplants. A total of 86.7% of LURD transplants were spousal transplants. A total of 29% of the patients who received LRDs were human leukocyte antigen-identical with their donors and 53% were haploidentical, versus 0 human leukocyte antigen-identical or haploidentical in the LURD group. RESULTS: Twenty-seven (90%) Of 30 LURD recipients are alive, as are 240 (86%) of 279 LRD recipients. Mean current creatinine is 1.6 mg/dl for the LURD group and 1.7 mg/dl for the LRD group Kaplan-Meier 1- and 5-year graft survival was 94.9% and 82.9% for the LRD group, 93.1% and 85.9% for the LURD group (p = not significant), and 84.6% and 70.7% for the cadaveric renal donor group (p < 0.05). CONCLUSIONS: LURD patient and graft survival is comparable to LRD transplants despite inferior human leukocyte antigen matching. LURD transplant survival is superior to that of cadaveric renal donor transplants. LURDs are an excellent but underused source of organs for renal transplant recipients.

Intramolecular mimicry. Identification and analysis of two cross-reactive T cell epitopes within a single protein.

The recognition of peptide Ags by T cells through the TCR has exquisite specificity. Cross-reactive T cell responses have been described; however, the structural basis for these responses is not known. We show that two peptides derived from the same protein can exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive T cell responses. In addition, we explore the structural basis for cross-reactivity. T cell hybridomas recognizing PiM and PiZ allelic forms of human alpha 1-antitrypsin (hAAT) each recognized both PiM 205-220 and PiM 335-350. These two peptides possessed primary sequence identity at only two of 16 amino acid residues. Cross-reactive peptides also exhibited homology at the bulk T cell level because lymph node T cells primed with one peptide proliferated to the other peptide in vitro. Critical amino acids for the responding T cells were determined, and the core was transferred into the less reactive peptide in an attempt to increase homology by increasing sequence identity. Interestingly, as identity increased, homology decreased: peptides with the least primary sequence identity appeared most homologous to the T cells. These results have important implications for understanding the development of autoimmune diseases, and imply that minimal obvious primary sequence identity may be sufficient to initiate cross-reactive T cell responses. The ability of structurally dissimilar peptides to mimic each other when bound to a class II MHC molecule may also be important to the understanding of T development and autoimmunity.

Regulation of the costimulator B7, not class II major histocompatibility complex, restricts the ability of murine kidney tubule cells to stimulate CD4+ T cells.

The proximal segment of murine kidney tubule cells (KTC) constitutively expresses low levels of class II major histocompatibility complex (MHC) that are upregulated during local and systemic inflammation. It is not known if KTC also express the costimulator molecules necessary for them to productively participate in immune responses and stimulate T cells. To answer this question, we studied the ability of KTC to present antigens to four Th1 clones. KTC did not induce T cell proliferation to specific antigen, superantigen, or concanavalin A. However, T cell receptors did engage the peptide/MHC ligand presented by KTC, as indicated by T cell enlargement and upregulation of interleukin-2 receptor expression. Importantly, KTC failed to express the Th1 costimulator, B7, as detected by fluorescence cytometry and reverse transcription polymerase chain reaction. We directly demonstrated that lack of B7 expression accounted for at least part of the KTC presentation defect, in that a KTC line transfected with the cDNA for B7 stimulated T cell proliferation to antigen. Our results suggest that epithelial cells expressing class II MHC have developed mechanisms to prevent costimulator expression and limit parenchymal tissue destruction. Failure of class II-expressing epithelial cells to limit costimulator expression may be an important component of organ-specific autoimmunity.

Tolerance to self and the processing and presentation of self antigens.

Antigen processing and presentation is critical to the generation and maintenance of self tolerance. The hemoglobin system has provided important data on self antigen processing and presentation in vivo. Hemoglobin/Ia complexes were detectable in the thymus before the time of positive and negative selection. In addition, thymic epithelial cells were shown to lack the costimulatory factors necessary to trigger T cell clone proliferation. We have extended these findings to the renal proximal tubule. This class II MHC-expressing epithelial cell was demonstrated to process and present foreign as well as self antigens to T cell hybridomas. Current studies are examining whether this epithelial cell possesses the costimulatory factors required to fully stimulate T cell clones, or whether the proximal tubule may play an important role in the maintenance of self tolerance. In addition we describe the exciting model of murine autoimmune myocarditis. We have demonstrated that this is a T cell mediated disease and believe that cardiac antigen presenting cells constitutively process and present the inciting self antigen, myosin. These studies may provide important insights into autoimmunity and self tolerance.

Processing and presentation of self and foreign antigens by the renal proximal tubule.

The renal proximal tubule (PT) in many ways resembles an APC. The PT is one of the few epithelial cells in the body reported to constitutively express the class II MHC molecules required to present Ag to CD4+ T cells. We questioned whether the PT could function as an APC in vitro and in vivo. Fluorescence cytometry demonstrated that the normal CBA/J PT constitutively expressed low levels of class II MHC and that this expression was markedly augmented by either IFN-gamma or systemic Listeria monocytogenes infection. Functionally, the PT from normal CBA/J mice also stimulated T cell hybridomas when cultured in vitro with Ag, and this ability was markedly up-regulated by both IFN-gamma as well as L. monocytogenes infection. To prove that the PT constitutively processed and presented self Ag in vivo, freshly isolated PT from mice transgenic for human alpha 1-antitrypsin were cultured with the appropriate T cell hybridoma in the absence of exogenous Ag. Strong stimulation of the T cell hybridoma occurred. Our data show that the renal proximal tubule processes and presents foreign Ag both in vitro and in vivo, and that it constitutively processes and presents the self Ag hAAT in vivo. These results have important implications for the understanding of renal interstitial autoimmune diseases as well as the interstitial nephritis that occurs in response to foreign Ag.

The processing and presentation of the self-antigen hemoglobin. Self-reactivity can be limited by antigen availability and costimulator expression.

APC do not distinguish between self- and foreign proteins. Previous studies from our laboratory demonstrated that most endogenous host APC constitutively processed and presented the self-Ag, hemoglobin (Hb), as detected by the Hb-specific T cell hybridoma, YO1.6. We have now examined APC in organs known to be involved in RBC degradation (liver Kupffer cells and splenic small resting B cells) for the presence of Hb/Ia complexes and for the expression of the costimulation necessary to trigger proliferation of T cell clones. We detected Hb/Ia complexes not only on splenic small resting B cells, but also on liver Kupffer cells. Interestingly, complexes were not present on lymph node small resting B cells. Splenic small resting B cells expressed costimulatory activity and efficiently stimulated the Th2 clones only. The opposite pattern was observed with liver Kupffer cells, which expressed costimulatory activity for Th1 clones only. However, if costimulatory activity was provided for the Th2 clones (IL-1 beta) and Th1 clones (allogenic spleen cells), the clones did proliferate in response to Kupffer cells and small resting B cells, respectively. In this report we have demonstrated that 1) endogenously formed self Hb/Ia complexes are expressed on splenic small resting B cells and liver Kupffer cells but not on lymph node small resting B cells and 2) these APC are also able to limit the expression of costimulatory activity for Th2 and Th1 T cell clones. Thus, endogenous APC not only constitutively process and present the self-Ag Hb, but also limit expression of the costimulatory activity necessary to trigger T cell proliferation against a self-Ag. The constitutive processing and presentation of self-Ag, as well as the regulation of costimulatory activity on APC, is likely an important feature of the maintenance of self-tolerance.

Vitamin D metabolites stimulate phosphatidylcholine transfer to renal brush-border membranes.

The phosphatidylcholine content of both the intestinal and renal brush-border membranes and ion transport are affected by 1,25-dihydroxycholecalciferol (1,25(OH)2D3). To investigate the mechanism of this effect, liposomes were prepared containing self-quenching concentrations of fluorescent phospholipid derivatives. When these liposomes were incubated with rat renal brush-border membrane vesicles, an immediate increase in the relative fluorescence of N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylcholine (NBD-PC) was detected, indicating transfer of NBD-PC into a non-quenched membrane. Addition of 1,25(OH)2D3 to the liposomes produced a dose-dependent stimulation of NBD-PC transfer to the acceptor brush-border membrane vesicles. Peripheral fluorescence was visible when the brush-border membrane vesicles were viewed with a fluorescent microscope. Using brush-border membrane vesicles from kidneys of vitamin D-deficient animals, quantitation of lipid transfer revealed a 1,25(OH)2D3 (10(-7) M) stimulation of NBD-PC transfer from 1.38 +/- 0.27 to 2.07 +/- 0.26 micrograms/h, and of PC transfer, assessed by vesicle phosphatidylcholine content, from 49.7 +/- 12 to 57.3 +/- 12 micrograms/mg protein per h (P less than 0.05). There was no significant transfer of N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In the absence of hormone, the amount of NBD-PC transferred to brush-border membrane vesicles prepared from normal rats was significantly greater than that transferred to brush-border membrane vesicles prepared from vitamin D-deficient animals (2.12 +/- 0.02 vs. 1.39 +/- 0.27 micrograms of NBD-PC/h, P less than 0.05). Both physiologic and pharmacologic concentrations of 1,25(OH)2D3 stimulated NBD-PC transfer with maximum response at 10(-14) M (2.98 +/- 0.15 micrograms/h). 24,25-Dihydroxycholecalciferol and 25-hydroxycholecalciferol (25(OH)D3) also stimulated transfer, although dose-response curves were less effective than for 1,25(OH)2D3. Cortisol and vitamin D-3 did not stimulate transfer. 1,25(OH)2D3 did not stimulate NBD-PC transfer between liposome populations.