Radiofrequency ablation for dysplasia in Barrett's esophagus restores ß-catenin activation within esophageal progenitor cells.

BACKGROUND AND AIMS: Endoscopic therapies for Barrett's esophagus (BE) associated dysplasia, particularly radiofrequency ablation (RFA), are popular alternatives to surgery. The effect of such therapies on dysplastic stem/progenitor cells (SPC) is unknown. Recent studies suggest that AKT phosphorylation of ß-Catenin occurs in SPCs and may be a marker of activated SPCs. We evaluate the effect of RFA in restoring AKT-mediated ß-Catenin signaling in regenerative epithelium. METHODS: Biopsies were taken from squamous, non-dysplastic BE, dysplastic BE and esophageal adenocarcinoma (EAC). Also, post-RFA, biopsies of endoscopically normal appearing neosquamous epithelium were taken at 3, 6, and 12 months after successful RFA. Immunohistochemistry and Western blot analysis was performed for Pß-Catenin(552) (Akt-mediated phosphorylation of ß-Catenin), Ki-67 and p53. RESULTS: There was no difference in Pß-Catenin552 in squamous, GERD, small bowel and non-dysplastic BE. There was a fivefold increase in Pß-Catenin(552) in dysplasia and EAC compared to non-dysplastic BE (P < 0.05). Also, there was a persistent threefold increase in Pß-Catenin(552) in neosquamous epithelium 3 months after RFA compared to native squamous epithelium (P < 0.05) that correlated with increased Ki-67. Six months after RFA, Pß-Catenin(552) and Ki-67 are similar to native squamous epithelium. CONCLUSIONS: Enhanced AKT-mediated ß-Catenin activation is seen in BE-associated carcinogenesis. Three months after RFA, squamous epithelial growth from SPC populations exhibited increased levels of Pß-Catenin(552). This epithelial response becomes quiescent at 6 months after RFA. These data suggest that elevated Pß-Catenin(552) after RFA denotes a repair response in the neosquamous epithelium 3 months post-RFA.

Divergent roles for p55 and p75 tumor necrosis factor receptors in the pathogenesis of MOG(35-55)-induced experimental autoimmune encephalomyelitis.

To clarify the role of tumor necrosis factor (TNF) in the inflammatory aspects of autoimmunity vs its potential role in the apoptotic elimination of autoreactive effector cells, we assessed the roles of the p55 (TNFR1/Tnfrsf1a/CD120a) and p75 (TNFR2/Tnfrsf1b/CD120b) TNF receptors in the pathogenesis of MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE). TNFR p55/p75(-/-) double knockout mice were completely resistant to clinical disease. TNFR p55(-/-) single knockout mice were also totally resistant to EAE, exhibiting reduced MOG(35-55)- specific proliferative responses and Th1 cytokine production, despite displaying equivalent DTH responses. Importantly, IL-5 was significantly increased in p55(-/-) mice. In contrast, p75(-/-) knockout mice exhibited exacerbated EAE, enhanced Th1 cytokine production, and enhanced CD4(+) and F4/80(+) CNS infiltration. Thus, p55/TNFR1 is required for the initiation of pathologic disease, whereas p75/TNFR2 may be important in regulating the immune response. These results have important implications for therapies targeting p55 and p75 receptors for treating autoimmune diseases.

Oral administration of avian tumor necrosis factor antibodies effectively treats experimental colitis in rats.

Tumor necrosis factor (TNF) is implicated in the pathogenesis of inflammatory bowel disease. Clinical trials indicate that intravenous infusion of anti-TNF antibody is an effective therapy for Crohn's disease. An oral anti-TNF therapy may be a preferred approach, reducing systemic side effects and eliminating the inconvenience and expense of administering infusions. We tested oral avian anti-TNF antibodies in the acute and chronic phases of a rodent colitis model. Efficacy was compared to sulfasalazine and dexamethsone. Rats with chemically induced colitis were treated orally with anti-TNF antibody, placebo, or comparator. Efficacy was assessed by change in colonic weight, morphology, histology, and tissue myeloperoxidase activity. Oral anti-TNF antibody, in both the acute and chronic phases of the model, significantly decreased all inflammatory end points and proved to be more effective than sulfasalazine and dexamethasone. Oral delivery of avian anti-TNF antibodies is an effective treatment of experimental colitis and may provide advantages to current parenteral anti-TNF antibodies.

Activation and peripheral expansion of murine T-cell receptor gamma delta intraepithelial lymphocytes.

BACKGROUND & AIMS: The intestinal epithelial compartment is populated by CD8(+) alpha beta and gamma delta intraepithelial lymphocytes (IELs), which monitor the integrity of the epithelial barrier. alpha beta IELs are activated by peptide antigens presented by class I major histocompatibility complex (MHC) molecules, but it is unclear how gamma delta IELs are activated. METHODS: G8 T-cell receptor (TCR) gamma delta transgenic (Tg) mice (specific for the class I MHC alloantigen, T22/10(b)) were crossed to class I MHC-deficient beta2-microglobulin-knockout (beta2m degrees) mice, and Tg+ IELs were examined for relative yields and surface and functional phenotype. RESULTS: Evidence for class I MHC-induced activation of Tg+ IELs was supported by the detection of 4-fold greater numbers of Tg+ IELs in G8 x beta2m+ mice that proliferated at 15-fold higher levels than IELs from G8 x beta2m degrees mice. However, expression of CD69, production of cytokine (interleukin 2 and interferon gamma), and detection of cytolytic function for IELs in G8 x beta2m degrees mice suggested that class I MHC was not required for gamma delta IEL development or maturation. CONCLUSIONS: These results suggest that CD8(+) TCR gamma delta IELs do not require class I MHC for development but support the notion that antigens presented by class I MHC molecules are involved in the peripheral expansion and differentiation of this subset.

Instrumented measurement of the posterolateral corner.

A new device called the Lars Rotational Laxiometer (Lars Inc, Dijon, France) is introduced to aid in the diagnosis of posterolateral rotatory instability of the knee. This device assigns a quantitative value for tibial external rotation. Three examiners each evaluated a separate group of 30 different subjects (total 180 knees) to obtain side-to-side differences. The subjects had no history of injury, pain, or instability. An external rotation measurement was performed at 30 degrees and 90 degrees of knee flexion. At 90 degrees, the mean side-to-side difference was 4.4 degrees (range, 3.7 degrees to 5.1 degrees); at 30 degrees it was 5.5 degrees (range, 4.7 degrees to 6.3 degrees). There was no significant difference with gender or age. The purpose of this study is to establish baseline side-to-side values for the posterolateral complex in normal knees. Objective values are obtainable with the Laxiometer.

Increased threshold for TCR-mediated signaling controls self reactivity of intraepithelial lymphocytes.

To examine the effect of self Ag on activation requirements of TCR-alphabeta intestinal intraepithelial lymphocytes (IELs), we utilized the 2C transgenic (Tg) mouse model specific for a peptide self Ag presented by class I MHC, H-2Ld. CD8alpha alpha and CD4-CD8- IELs from syngeneic (H-2b, self Ag-) and self Ag-bearing (H-2b/d, self Ag+) strains were examined for their ability to respond in vitro to P815 (H-2d) cell lines expressing the endogenous antigenic peptide, p2Ca. Proliferation, cytokine production, and CTL activity were elicited in IEL T cells isolated from self Ag- H-2b mice when stimulated with P815 cells expressing basal levels of self Ag. These responses were enhanced following the addition of exogenous p2Ca peptide and ectopic expression of the costimulatory molecule, B7-1. By comparison, IEL from self Ag-bearing mice failed to respond to basal levels of self Ag presented by P815 cells even in the presence of B7-1-mediated costimulation. However, the addition of increasing amounts of exogenous p2Ca peptide induced a response from the in vivo "tolerized" T cells. These results suggest that exposure to self Ag in vivo increased the threshold of TCR activation of Ag-exposed self-reactive IELs. The dependence of increased signal 1 to activate self-reactive IELs suggests a defect in TCR signaling that may maintain self tolerance in vivo. These data suggest that conditions that overcome signal 1 IEL defects may initiate autoreactive responses in the intestine.

IFN-gamma-activated primary murine astrocytes express B7 costimulatory molecules and prime naive antigen-specific T cells.

Astrocytes may serve as effectual APCs for T cell-mediated immune responses to myelin components during multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Although astrocytes have been reported not to constitutively express MHC class II molecules, expression is up-regulated during active EAE and by in vitro incubation with IFN-gamma. Previous studies have reported that cytokine-activated astrocytes are able to activate Ag-specific previously activated T cells, but not naive alloreactive T cells. In the current study, we show that a subset of primary murine astrocytes constitutively expresses B7-2 molecules, as determined by FACS and PCR analyses, and up-regulates surface expression and mRNA levels of both B7-2 and B7-1 upon IFN-gamma stimulation. In contrast to earlier reports, we found that both untreated and IFN-gamma-treated astrocytes were able to stimulate proliferation of previously activated OVA-specific Th1 cells. In contrast, only IFN-gamma-treated astrocytes activated naive, transgenic OVA-specific T cells. Astrocyte-induced activation of both OVA-specific naive T cells and activated Th1 cells was dependent primarily on B7-2-mediated costimulation, as proliferation was inhibited by CTLA4-Ig and by anti-B7-2 mAbs. These results suggest that astrocytes in an inflammatory environment have the capacity to express the required MHC class II and B7 costimulatory molecules necessary for efficient activation of naive T cells. Since we have shown that T cells specific for endogenous myelin epitopes released during acute EAE play the major pathologic effector role in subsequent disease relapses (epitope spreading), astrocytes could play a role in the local activation and expansion of these responses.

The predictive value of intraoperative KT-1000 arthrometer measurements in single incision anterior cruciate ligament reconstruction.

We reviewed 28 patients who underwent anterior cruciate ligament reconstruction with immediate, 1-, 2-, and 3-year postreconstruction KT-1000 manual maximum testing. Arthrometer measurements were correlated with functional knee criteria to evaluate the ability of the KT-1000 to predict postreconstruction functional results. Despite a range of immediate postreconstruction arthrometer injured-minus-normal (I - N) differences, there was no association with I - N difference at last follow-up. Patients followed-up for 1 year were not different from those who were followed-up for longer with respect to intraoperative or 1-year I - N difference or functional performance scores. Furthermore, excellent functional knee scores were the norm at all stages of follow-up despite a wide range of arthrometric laxity changes. The results suggest that functional knee criteria, although partially subjective, are more useful indicators of outcome than intrareconstruction and postreconstruction arthrometric measures.

Lethal murine graft-versus-host disease induced by donor gamma/delta expressing T cells with specificity for host nonclassical major histocompatibility complex class Ib antigens.

Although T-cell receptor (TCR) alpha/beta expressing cells have a well-known role in graft-versus-host disease (GVHD) generation, the role of TCR gamma/delta expressing cells in this process has remained unclear. To elucidate the potential function of TCR gamma/delta cells in GVHD, we have used transgenic (Tg) H-2d mice (termed G8) that express gamma/delta heterodimers on a high proportion of peripheral T cells. In vitro, G8 Tg gamma/delta T cells proliferate to and kill C57BL/6 (B6) (H-2b) which express gene products (T10b and T22b) from the nonclassical major histocompatibility complex (MHC) class Ib H-2T region. The infusion of G8 Tg (H-2Td) TCR gamma/delta cells into lethally irradiated [900 cGy total body irradiation (TBI)] B6 (H-2b) mice resulted in the generation of lethal GVHD characterized histologically by destruction of the spleen, liver, lung, and colon. Lethal GVHD was prevented by the injection of anti-TCR gamma/delta monoclonal antibodies. Immunohistochemical analysis of B6 recipients post-bone marrow transplantation (BMT) confirmed that G8 Tg TCR gamma/delta cells infiltrated GVHD target tissues (skin, liver, colon, and lung) and were absent in recipients treated with anti-TCR gamma/delta monoclonal antibodies (MoAbs) but not anti-CD4 plus anti-CD8 MoAbs. In contrast, injection of TCR gamma/delta+ cells into irradiated (900 cGy TBI) B6.A-TIaa BoyEg mice that do not express either T10b or T22b did not induce lethal GVHD. Similarly, in a different GVHD system in which sublethal irradiation without bone marrow (BM) rescue was used, B6 but not B6.A-TIaa/BoyEg mice were found to be susceptible to TCR gamma delta+ cell mediated GVHD-induced lethality characterized by an aplasia syndrome. These results demonstrate that TCR gamma/delta cells have the capacity to cause acute lethal GVHD in mice and suggest that nonclassical MHC class Ib gene products expressed on GVHD target organs are responsible for G8 Tg TCR gamma/delta+ cell mediated lethality.

CD28-mediated costimulation is necessary for the activation of T cell receptor-gamma delta+ T lymphocytes.

The role of costimulation in the activation of TCR-gamma delta cells in normal mice and mice transgenic (tg) for a TCR-gamma delta receptor was investigated. Activation of TCR-gamma delta cells required two signals. One signal was mediated by TCR occupancy, whereas a second signal was provided by accessory cells. The importance of the CD28/B7 interaction in the delivery of the second signal was demonstrated in multiple ways. First, addition of a soluble fusion protein homolog of CD28, CTLA4Ig, significantly inhibited the activation of G8 tg splenic TCR-gamma delta lymphocytes and intestinal epithelial TCR-gamma delta lymphocytes by Ag-bearing lymphocytes during primary stimulation. Similarly, both proliferation and IFN-gamma production were inhibited by addition of CTLA4Ig to secondary antigenic stimulation of G8 tg TCR-gamma delta cells. Second, an Ag-bearing thymoma, EL-4, was only able to stimulate expanded G8 tg TCR-gamma delta cells when the thymoma expressed B7. This stimulation was blocked by both CTLA4Ig and anti-B7 antibody. Third, antibodies to CD28 were able to mimic the costimulatory affect of APC. TCR-gamma delta cells cultured with either Ag-bearing fixed stimulator cells or submitogenic concentrations of immobilized anti-pan TCR-gamma delta mAb proliferated only in the presence of anti-CD28 mAb. Finally, G8 tg cells produced IL-2 only in the presence of APC costimulation or anti-CD28 antibodies, and the addition of exogenous rIL-2 overcame the need for costimulation. Thus, autocrine IL-2 production is one of the major consequences of TCR-gamma delta cell costimulation. Together these data demonstrate that costimulation is necessary for the activation of TCR-gamma delta cells and can occur through CD28 interaction.

Control of self-reactivity in the intestine.

In the intestine maintenance of self-tolerance may involve tissue-specific self-Ags, APCs, 'second signals', and extrathymic pathways of T cell maturation. These factors combine to create a unique environment where autoimmune tissue destruction is prevented despite local inflammatory influences. In this review we summarize our findings using a TCR-gamma delta transgenic model where self-tolerance was maintained by clonal deletion for cells localizing to peripheral lymphoid tissue and by clonal anergy for cells localizing to the intraepithelial compartments. Several possible explanations exist for these results but in general, these findings have implications for the maintenance of self-tolerance of normal TCR-alpha beta and TCR-gamma delta IELs in epithelial tissues such as the intestine.

Chemotactic peptide effects on intestinal electrolyte transport.

The bacterial-derived chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) increases short-circuit current (Isc) and arachidonic acid metabolism (AAM) in rabbit ileum and distal colon. Serosal (s) or mucosal (m) addition of fMLP transiently increases Isc. Half-maximally effective dose and maximal increases in Isc were 32 nM and 84 microA/cm2 in ileum and 234 nM and 80 microA/cm2 in colon, respectively. Piroxicam, a cyclooxygenase inhibitor, diminished the Isc response by 97% in colon and 69% in ileum. Changes in Isc were dependent on Cl- and HCO3- in the bathing media. In ileum, fMLP inhibited m-to-s 36Cl- fluxes and stimulated s-to-m 36Cl- fluxes. These changes in Cl- flux were also inhibited by piroxicam. fMLP stimulated prostaglandin E2 (PGE2) release in intact tissue and in isolated subepithelial components. Increased tissue adenosine 3',5'-cyclic monophosphate levels were detected in intact tissue but not in isolated components. Previous desensitization of ileum to PGE1 inhibited fMLP stimulation of Isc in ileum by 88%. Desensitization to fMLP in ileum failed to alter the effect of PGE1 (10 microM) on Isc. In isolated microsomal membranes of ileal enterocytes, fMLP binding sites could not be demonstrated, suggesting that fMLPs action was initially mediated via stimulation of nonepithelial cell cyclooxygenase activity. The above results indicate that fMLP stimulates net secretion in both ileum and colon probably by the activation of AAM.