Constitutively active CCK2 receptor splice variant increases Src-dependent HIF-1 alpha expression and tumor growth.

Gastrointestinal (GI) cancers ectopically express multiple splice variants of the cholecystokinin-2 (CCK(2))/gastrin receptor; however, their relative contributions to the cancer phenotype are unknown. The aim of this study was to compare the effects of CCK(2) receptor (CCK(2)R) and CCK(2i4sv)R expression on cell growth both in vitro and in vivo using a human epithelial cell model, HEK239. In vitro, receptor variant expression did not affect cell proliferation either in the absence or presence of agonist. However, in vivo, the expression of CCK(2i4sv)R, but not CCK(2)R, increases HEK293 tumor growth in a constitutive, Src-dependent manner. Enhanced tumorigenicity of CCK(2i4sv)R is associated with an Src-dependent increase in the transcription factor, hypoxia-inducible factor-1alpha, its downstream target, vascular endothelial growth factor and tumor micro-vessel density, suggesting that CCK(2i4sv)R may contribute to the growth and spread of GI cancers through agonist-independent mechanisms that enhance tumor angiogenesis.

HLA-DQ6 transgenic mice resistance to experimental autoimmune myasthenia gravis is linked to reduced acetylcholine receptor-specific IFN-gamma, IL-2 and IL-10 production.

To comprehend the reduced susceptibility of HLA-DQ6 transgenic mice in comparison with HLA-DQ8 mice, to experimental autoimmune myasthenia gravis (EAMG), we immunized them with acetylcholine receptor (AChR) and examined in vitro, the proliferative and cytokine responses to AChR. When immunized with AChR and examined for AChR-specific lymphocyte responses to AChR, EAMG-resistant DQ6 mice exhibited significantly reduced in vitro lymphoproliferative and cytokine responses to AChR, compared to DQ8 mice. The differences in susceptibility were not linked to a difference in peptide recognition by AChR-specific lymphocytes. AChR T cell epitope mapping showed that both DQ6 and DQ8 responded to the same epitopes, although to varying degrees. Resistance of DQ6 transgenic mice to EAMG was linked to a dramatic suppression of AChR-specific IFN-gamma, IL-2 and IL-10 productions by AChR-primed lymph node cells.

Fas/Fas ligand pathway, apoptosis, and clonal anergy involved in systemic acetylcholine receptor T cell epitope tolerance.

The cellular mechanisms of high dose systemic acetylcholine receptor (AChR) T cell epitope, alpha 146--162 peptide-induced tolerance in experimental myasthenia gravis were examined. CD4 cells are the prime target for alpha 146--162 peptide-induced tolerance. The expression of CD69, Fas, and B7.2 molecules on AChR-immune lymphocytes was enhanced within 4--12 h after tolerance induction. A high dose of alpha 146--162 peptide in IFA failed to suppress T cell proliferation and/or clinical myasthenia gravis in lpr and gld mice deficient in Fas and Fas ligand, respectively. A high dose of alpha 146--162 peptide in IFA in AChR-immunized mice induced apoptosis of BV6 cells. Further, reconstitution of IL-2 in vitro-recovered alpha 146--162 peptide tolerized T cell proliferation, IFN-gamma, and IL-10 production. The findings implicate the possible role of Fas-/Fas ligand-mediated apoptosis and the resulting clonal anergy as the mechanisms of high dose AChR alpha 146--162 peptide-induced tolerance on CD4 cells.

Lymphotoxin-alpha deficiency completely protects C57BL/6 mice from developing clinical experimental autoimmune myasthenia gravis.

A complete prevention of clinical experimental autoimmune myasthenia gravis (EAMG) was observed in lymphotoxin (LT)-alpha deficient (LT-alpha(-/-)) mice compared to LT-alpha(+/+) mice when immunized with acetylcholine receptor. However, only a partial prevention of clinical EAMG incidence was observed in LT-beta(-/-) mice compared to LT-beta(+/+) mice. LT-alpha(-/-)and LT-beta(-/-) mice had lower mean titers of total IgG, IgG(1), IgG(2a) and IgG(2b) and higher or equal mean titers of IgM anti-AChR antibodies compared to controls. Therefore, LT-alpha(-/-)and LT-beta(-/-) AChR immunized mice are capable of mounting a primary (IgM) humoral immune response to AChR, but are less capable of switching to the pathogenic anti-AChR IgG isotypes. LT could play a significant role in the pathogenesis of myasthenia gravis.

Experimental autoimmune myasthenia gravis in the mouse.

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or eel. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography, as described here. In addition, EAMG is frequently characterized by the presence of antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content.

Suppression of experimental autoimmune myasthenia gravis in IL-10 gene-disrupted mice is associated with reduced B cells and serum cytotoxicity on mouse cell line expressing AChR.

To analyze the role of interleukin-10 (IL-10) in experimental autoimmune myasthenia gravis (EAMG) pathogenesis, we induced clinical EAMG in C57BL/6 and IL-10 gene-knockout (KO) mice. IL-10 KO mice had a lower incidence and severity of EAMG, with less muscle acetylcholine receptor (AChR) loss. AChR-immunized IL-10 KO mice showed a significantly higher AChR-specific proliferative response, altered cytokine response, lower number of class II-positive cells and B-cells, but a greater CD5(+)CD19(+) population than C57BL/6 mice. The lower clinical incidence in IL-10 KO could be explained not by a reduction of the quantity, but by a possible difference in the pathogenicity of anti-AChR antibodies.

The effect of B cell deficiency on the immune response to acetylcholine receptor and the development of experimental autoimmune myasthenia gravis.

To study the involvement of B cells in the immune response to acetylcholine receptor (AChR), B-cell-deficient (mu mutant) and control wild-type C57BL/6 mice were immunized with AChR and assessed for clinical and immunopathological manifestations of experimental autoimmune myasthenia gravis (EAMG). The mu mutant mice failed to generate anti-AChR antibodies and were completely resistant to the induction of EAMG. However, mu mutant mice developed clinical EAMG when antibodies to the AChR main immunogenic region were passively transferred. Further, the in vivo expansion of lymph node cells after AChR immunization was greatly impaired in mu mutant mice. The mu mutant mice gave an effective in vitro T cell immune response to the immunodominant pathogenic AChR alpha chain peptide 146-162 (alpha 146-162) and to the whole AChR protein when tested on day 90 after immunization with AChR, whereas the response to both AChR and its alpha 146-162 peptide was reduced when tested on day 7 after immunization. The in vitro production of IFN-gamma and IL-2 by AChR-specific and alpha 146-162 peptide-specific lymphocytes was lower in mu mutant mice. The AChR immune mu mutant T cells proliferated and produced IFN-gamma when AChR or alpha 146-162 peptide was presented by wild-type irradiated AChR-primed antigen-presenting cells (APCs). This indicates that B cells are important in the processing and presentation of AChR dominant peptide in vitro during the initial immune response to AChR. However, APCs of non-B-cell lineage are sufficient to process AChR and prime the T cells to AChR dominant T cell epitope peptides.

Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis.

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.

Experimental autoimmune myasthenia gravis in B10.BV8S2 transgenic mice: preferential usage of TCRAV1 gene by lymphocytes responding to acetylcholine receptor.

Multiple TCRBV genes have been implicated in experimental autoimmune myasthenia gravis (EAMG) pathogenesis in susceptible H-2(b) strains of mice. We studied the contribution of specific TCRBV and AV genes in EAMG pathogenesis using B10.BV8S2 transgenic mice (H-2[b]). The TCR transgenic mice predominantly have TCRBV8S2 transgene, but can use any of the endogenous AV gene repertoire. The transgenic mice were immunized with acetylcholine receptor (AChR) in CFA and evaluated for EAMG pathogenesis. Although the lymphocyte responses to AChR in B10.BV8S2 transgenic and nontransgenic TCR wild-type mice were equivalent, a marked reduction in lymphocyte response to the dominant AChR alpha chain peptide 146-162 was observed in the TCR transgenic mice. After boosting with AChR in CFA, anti-AChR Abs were detected in the serum, and 14 of 42 (33%) of the TCR transgenic mice developed clinical EAMG. Furthermore, EAMG in TCR transgenic mice was prevented by treatment with mAb to TCRBV8, which depleted BV8-expressing T cells. Cloning and sequencing of TCRAV genes from AChR-reactive T cells from B10.BV8S2 transgenic mice revealed a pattern of restricted TCRAV gene usage. The majority (60%) of the clones sequenced showed a sequence identical with that of the TCRAV1S8 gene. In the normal spleen cells of TCR transgenic mice, AV gene usage was more random. Thus, despite the presence of a complete endogenous TCRAV repertoire in B10.BV8S2 transgenic mice, T cells responding to AChR preferentially used a single endogenous TCRAV gene, thus implicating the involvement of the TCRAV1S8 gene in EAMG pathogenesis.

IFN-alpha therapy is effective in suppressing the clinical experimental myasthenia gravis.

To study the therapeutic efficacy of IFN-alpha after the onset of clinical signs of experimental autoimmune myasthenia gravis (EAMG), we treated mice with clinical EAMG with recombinant human IFN-alpha or mouse IFN-alpha. In the first experiment, 7 of 16 (44%) mice had a complete clinical remission in the recombinant human IFN-alpha-treated group, in contrast to none in the placebo group (0/14) (p = 0.006). There was a higher incidence of death and severe disease in the placebo group (7/14) relative to the IFN-alpha group (4/16). In the second experiment, 6 of 18 (33%) mice in the mouse IFN-alpha-treated group had a complete clinical remission, while none of 17 (0%) mice in the placebo-treated group had remission (p = 0.011). Again, more mice died or worsened in the placebo group (11/17) compared with the IFN-alpha group (7/18). IFN-alpha treatment significantly reduced the anti-acetylcholine receptor (AChR) Ab levels, especially the IgG1 and IgG2b isotypes, and the amount of anti-AChR Abs bound to muscle AChR. IFN-alpha treatment also lowered CD4 cells in the lymph nodes and spleen, and suppressed the in vitro lymphocyte proliferative response to AChR and its dominant peptide in a dose-dependent manner.

Association of colony variation in Serratia marcescens with the differential expression of protease and type 1 fimbriae.

Several clinical isolates of Serratia marcescens were found to dissociate on peptone glycerol agar into colonies with red and pink or white and gray phenotypes that differ in the expression of proteolytic activity and mannose-sensitive type of hemagglutination. Colonies of red and white type were proteolytically active but did not express hemagglutination, whereas pink and gray colonies were protease-deficient but agglutinated guinea pig erythrocytes. Site-directed mutagenesis of a red laboratory strain S. marcescens SM6 resulted in selection of protease negative derivative prt::G7 which expressed the pink phenotype with hemagglutinating activity. It is suggested that a DNA-regulatory element may be involved in this type of colony variation.

IFN-alpha treatment suppresses the development of experimental autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an Ab-mediated autoimmune neuromuscular disease and is linked to MHC class II beta-chain polymorphism. Corticosteroids and azathioprine are the primary immunosuppressive drugs used in the treatment of MG. These drugs have significant side effects and have limited efficacy. Therefore, drugs with fewer side effects and greater efficacy are being sought. IFN-alpha is a potent immunomodulator and has been shown to down-regulate MHC class II expression on lymphoid cells. MHC class II expression is critical for the development of experimental autoimmune myasthenia gravis (EAMG). Because of the immunomodulating effects of IFN-alpha and its effect on the MHC class II expression, we tested the therapeutic efficacy of IFN-alpha on EAMG induced by immunization with acetylcholine receptor (AChR) in CFA. IFN-alpha (10(5) IU three times weekly for 5 wk) treatment started 1 wk after the second immunization with AChR in CFA, when autoimmunity to AChR is well established, reduced the incidence of clinical EAMG by more than 50% in two separate experiments (p = 0.04 and 0.008). Therefore, IFN-alpha could be a potential agent for the control of MG, and other Ab-mediated autoimmune diseases.

TCR gene usage in experimental autoimmune myasthenia gravis pathogenesis. Usage of multiple TCRBV genes in the H-2b strains.

Experimental autoimmune myasthenia gravis (EAMG) is an Ab-mediated autoimmune disease. The pathogenic auto-antibody production depends on the activation of CD4+ cells after their TCR interact with dominant T cell epitopes within acetylcholine receptor (AChR) in the context of the MHC class II molecule. In vitro analysis suggested that the TCRBV6 was the predominant TCR that recognized AChR and one of the dominant epitopes, alpha 146-162, in C57BL6 (B6, H-2b) mice. However, in vivo depletion of TCRBV6 cells in H-2b mice by anti-TCRBV6 mAb neither suppressed the in vitro immune response to AChR nor prevented development of EAMG. Moreover, B10.TCRc (H-2b) strain with a genomic deletion of TCRBV genes including TCRBV6, and B10.V beta 8.2 transgenic mice with a restricted TCRBV8S2 T cell repertoire, responded to AChR, alpha 146-162, and developed EAMG after immunizations with AChR/CFA. These data suggest that more than one TCRBV-bearing cell having the affinity for AChR-dominant peptides is involved in pathogenesis. Therefore, depletion of a single TCRBV (e.g., TCRBV6) with mAb may not be sufficient to completely suppress the response to AChR and development of EAMG. However, if a similar amino acid sequence in the TCR-VDJ (e.g., CDR3) region among different TCRBV gene(s) could be involved in recognizing the dominant AChR epitope(s), then motif-specific mAb reactive to the common motif within the VDJ region of different TCR could be used to eliminate the T cell clones involved in EAMG.

Effect of MHC class I and CD8 cell deficiency on experimental autoimmune myasthenia gravis pathogenesis.

MHC class I and CD8+ cell deficiency have either prevented systemic lupus erythematosus-like disease in mice or enhanced type I diabetes in nonobese diabetic mice. To study the involvement of MHC class I and class I-restricted CD8+ T cells in the induction of a classical Ab-mediated disease, experimental autoimmune myasthenia gravis (EAMG), we immunized beta 2 microglobulin (beta 2-m) gene-disrupted (beta 2 m-/-) C57BL10 (B10) mice, deficient in class I gene expression and CD8+ cells, and heterozygous (beta 2-m+/-) B10 mice with normal expression of class I molecules and sufficient CD8+ cells with Torpedo acetylcholine receptor in CFA, and assessed them for clinical and immunopathologic manifestations of EAMG. Despite MHC class I and CD8+ cell deficiency, beta 2-m-/- mice developed EAMG. Moreover, the incidence of EAMG in the beta 2-m-/- mice was higher than that of beta 2-m+/- heterozygous mice with normal class I expression and frequency of CD8+ cells. The finding provided direct genetic evidence against a pathogenic effector role in C57BL10 mice for MHC class I molecule and class I-restricted CD8+ T cells in EAMG pathogenesis.

The pathogenic role of acetylcholine receptor alpha chain epitope within alpha 146-162 in the development of experimental autoimmune myasthenia gravis in C57BL6 mice.

One of the dominant T cell epitopes in the acetylcholine receptor (AChR) alpha chain lies within the region 146-162 and has been implicated in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) in C57BL6 (B6) mice. To directly examine the pathogenic potential of alpha 146-162 in EAMG, B6 mice were primed with AChR in complete Freunds adjuvant (CFA) and subsequently boosted twice with either alpha 146-162 or a control peptide in CFA. Seventy percent of the mice boosted with alpha 146-162 developed muscle weakness characteristic of EAMG, while none of the mice boosted with the control peptide showed any clinical signs of the disease. Thus, the data provided evidence for epitope within AChR alpha 146-162 as one of the EAMG-inducing pathogenic epitopes in B6 mice.

Major histocompatibility complex class II gene disruption prevents experimental autoimmune myasthenia gravis.

To analyze the impact of lack of MHC class II gene expression, and to demonstrate the direct genetic evidence for the involvement of the MHC class II gene product in the development of experimental autoimmune myasthenia gravis (EAMG), MHC class II gene-disrupted C57BL6 mutant (-/-) and EAMG-susceptible MHC class II wild-type C57BL6 mice (+/+) were evaluated for the clinical and immunopathologic manifestations of EAMG. The deficiency of MHC class II, and therefore, CD4+ T cells, completely prevented the C57BL6 MHC class II mutant (-/-) mice from mounting an autoimmune response to the nicotinic acetylcholine receptor. Further, the mutant (-/-) mice failed to show any immunopathologic and clinical manifestations of EAMG. The data unequivocally provide direct genetic evidence for the essential role of MHC class II molecules in the induction of EAMG, and rule out any pathogenic effector role for MHC class I-restricted CD8+ T cells, gamma delta TCR-bearing cells, or NK cells, which are intact in the MHC class II mutant mice in the induction of EAMG.

[Trial application of transfection tests in feasibility studies for transforming DNA in white blood cells of patients with chronic lymphatic leukemia]. / Próba zastosowania testów transfekcyjnych w badaniach wlasciwosci transformujacych DNA z krwinek bialych chorych na przewlekla bialaczke limfatyczna.

Transfection technique with the use of high molecular DNA was applied for the investigations of peripheral blood white cells DNA transforming properties in patients with chronic lymphatic leukemia B. Using transfection tests in liquid medium (focus assay) and colony forming in soft agar, the differences in L(tk-) cells transformation were noted following an addition of neoplastic DNA. Medium collected over transformants has shown mitogen properties in micro-mitogen tests on cells BALB/c 3T3. No correlation between clinical stage of the disease and the results of tests was observed.