MHC class I-like MILL molecules are beta2-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression

MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta2-microglobulin (beta2m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta2m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.     

Enhanced immunogenicity of heat shock protein 70 peptide complexes from dendritic cell-tumor fusion cells

We have developed a molecular chaperone-based tumor vaccine that reverses the immune tolerance of cancer cells. Heat shock protein (HSP) 70 extracted from fusions of dendritic (DC) and tumor cells (HSP70.PC-F) possess superior properties such as stimulation of DC maturation and T cell proliferation over its counterpart from tumor cells. More importantly, immunization of mice with HSP70.PC-F resulted in a T cell-mediated immune response including significant increase of CD8 T cells and induction of the effector and memory T cells that was able to break T cell unresponsiveness to a nonmutated tumor Ag and provide protection of mice against challenge with tumor cells. By contrast, the immune response to vaccination with HSP70-PC derived from tumor cells is muted against such nonmutated tumor Ag. HSP70.PC-F complexes differed from those derived from tumor cells in a number of key manners, most notably, enhanced association with immunologic peptides. In addition, the molecular chaperone HSP90 was found to be associated with HSP70.PC-F as indicated by coimmunoprecipitation, suggesting ability to carry an increased repertoire of antigenic peptides by the two chaperones. Significantly, activation of DC by HSP70.PC-F was dependent on the presence of an intact MyD88 gene, suggesting a role for TLR signaling in DC activation and T cell stimulation. These experiments indicate that HSP70-peptide complexes (PC) derived from DC-tumor fusion cells have increased their immunogenicity and therefore constitute an improved formulation of chaperone protein-based tumor vaccine.     

Mechanical stretch upregulates IGFBP-1 secretion from decidualized endometrial stromal cells

Decidualization is an essential process of endometrial differentiation for embryo implantation and maintenance of pregnancy. Recently, uterine movement-induced mechanical stress was noticed to have possible effects on endometrial functions. In this study, we addressed the possible effect of mechanical stress on the process of decidualization of endometrial stromal cells (ESC). ESC were cultured on flexible-bottomed culture plates. After decidualization was achieved with estradiol and progesterone for 12 days, cultures were continued for 24 h with or without cyclic stretch (25% elongation) in serum-free conditions at a rate of 2 cycles/min using a computer-operated cell tension system. Concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1), a marker of decidualization, in the conditioned medium were measured by specific ELISA, and IGFBP-1 mRNA expression in the ESC was measured by RT-PCR. Cyclic stretch remarkably increased IGFBP-1 secretion from decidualized ESC. It also increased IGFBP-1 mRNA in decidualized ESC. The increase in IGFBP-1 secretion was inhibited by actinomycin D but not by indomethacin, PD-98059, or H-89. Conditioned medium of decidualized ESC cultured with cyclic stretch increased IGFBP-1 secretion from decidualized ESC cultured under stationary conditions. These findings imply that uterine movement modulates decidualization of the endometrium and has a regulatory effect on reproduction.     

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4⁺ and/or CD8⁺ T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4⁺ T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8⁺ T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4⁺ T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8⁺ T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4⁺ T cell-mediated antitumor activity in vivo.     

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.     

Immunization with heat shock protein 105-pulsed dendritic cells leads to tumor rejection in mice

Recently, we reported that heat shock protein 105 (HSP105) DNA vaccination induced anti-tumor immunity. In this study, we set up a preclinical study to investigate the usefulness of dendritic cells (DCs) pulsed with mouse HSP105 as a whole protein for cancer immunotherapy in vivo. The recombinant HSP105 did not induce DC maturation, and the mice vaccinated with HSP105-pulsed BM-DCs were markedly prevented from the growth of subcutaneous tumors, accompanied with a massive infiltration of both CD4+ T cells and CD8+ T cells into the tumors. In depletion experiments, we proved that both CD4+ T cells and CD8+ T cells play a crucial role in anti-tumor immunity. Both CD4+ T cells and CD8+ T cells specific to HSP105 were induced by stimulation with HSP105-pulsed DCs. As a result, vaccination of mice with BM-DCs pulsed with HSP105 itself could elicit a stronger tumor rejection in comparison to DNA vaccination.     

Gene expression profile in the heart of spontaneous dwarf rat: in vivo effects of growth hormone

Excess and deficit of growth hormone (GH) both affect cardiac architecture as well as its function. To date, experimental and clinical studies have reported that GH has an inotropic effect on animal and human heart, however, it remains controversial whether GH is applicable to the treatment for the patients with chronic heart failure. Also, the mechanism by which GH exerts these biological effects on the heart is not well understood. In this study, we attempted to specify the genes regulated by GH in the heart of spontaneous dwarf rat using a microarray analysis. We found that soluble forms of guanylate cyclase, cofilin1, and thymosin beta4 mRNA were up-regulated in the heart by GH treatment. On the other hand, acyl-CoA synthetase, aldosterone receptor, myosin regulatory light chain, troponin T, laminA, and beta-actin mRNA were down-regulated. These results suggest GH regulates essential molecules that regulate structural, contractile, remodeling, and regenerative functions. Collectively, our data indicate a new integrative understanding for the biological effects of GH on cardiac function.     

Substrate specificity of archaeon Sulfolobus tokodaii biotin protein ligase

Biotin protein ligase (BPL) is an enzyme mediating biotinylation of a specific lysine residue of the carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recently found that the substrate specificity of BPL from archaeon Sulfolobus tokodaii is totally different from those of many other organisms, in reflection of a difference in the local sequence of BCCP surrounding the canonical lysine residue. There is a conserved glycine residue in the biotin-binding site of Escherichia coli BPL, but this residue is replaced with alanine in S. tokodaii BPL. To test the notion that this substitution dictates the substrate specificity of the latter enzyme, this residue, Ala-43, was converted to glycine. The K(m) values of the resulting mutant, A43G, for substrates, were smaller than those of the wild type, suggesting that the residue in position 43 of BPL plays an important role in substrate binding.     

Elevated expression of ERK 2 in human tumor cells chronically treated with PD98059

We examined the effect of chronic exposure of tumor cells to a mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK) kinase inhibitor, PD98059, on cell proliferation was investigated. Human renal carcinoma cells (ACHN) and prostatic carcinoma cells (DU145) were cultured in the presence of PD98059 for more than 4 weeks (denoted ACHN (PD) cells and DU145 (PD) cells, respectively) and proliferation and signal transduction pathways were examined. PD98059 significantly inhibited the proliferation of parental cells. However, PD98059 failed to inhibit proliferation of ACHN (PD) and DU145 (PD) cells significantly. Expression of ERK 1 and 2 was elevated in these cells. These phenotypes were reversible. Downregulation of ERK 2, but not ERK 1, by small interfering RNA significantly inhibited the proliferation of ACHN (PD) and DU145 (PD) cells. Taken together, chronic exposure of tumor cells to PD98059 induced elevated expression of ERK 2, which was associated with decreased sensitivity of cellular proliferation to PD98059.     

Immunological effects of recombinant feline interferon-omega (KT-80) administration in the dog

The immunological effects of recombinant feline interferon-omega (rFeIFN-omega ; KT-80, Toray) were examined on administration to healthy dogs. The activities of whole blood cells, macrophages, and natural killer cells were enhanced. Moreover, the whole blood activity was examined when KT-80 was administered to dogs which had been diagnosed as having natural canine parvovirus (CPV) infection. Only some cases in which the activity increased until 3 hr post-administration survived. These results suggest that rFeIFN-omega (KT-80) treatment enhanced the cellular immunity of normal dogs, and could exert significant therapeutic effects on only natural CPV infected dogs with induced continuous immunoenhancement.