Expression Pattern of Tumor Necrosis Factor-α-Induced Protein 8-Like 2 in Acute Rejection of Cardiac Allograft.

BACKGROUND: Tumor necrosis factor-α-induced protein-8 like-2 (TIPE2) is a negative regulator of innate immunity and cellular immunity, yet the expression pattern of TIPE2 in acute rejection of cardiac allograft remain enigmatic. METHODS: We developed cardiac transplantation models and divided into 3 groups: a naive group, a syngeneic group, and an allogeneic group. Then, we detected the messenger RNA and protein of TIPE2 in cardiac allografts. Real-time polymerase chain reaction showed expression of CD4 and CD8 in the donor heart, and immunofluorescence assay revealed the association between T cells and TIPE2. RESULTS: In our study, we first found that the expression of TIPE2 in cardiac allografts is upregulated compared with the syngeneic control, and increases in a time-dependent manner. The immunocytochemistry of heart grafts revealed a strong expression of TIPE2 in the inflammatory cells, but not in the cardiomyocytes. Finally, we proved that CD4+ and CD8+ T cells infiltrated cardiac allografts abundantly, which express ample TIPE2. CONCLUSIONS: The upregulated expression of TIPE2 in cardiac allografts, mainly came from T cells, which infiltrated the donor heart. This finding indicates that there may be an association between TIPE2 and acute cardiac allograft rejection.

Changes in histone acetylation during oocyte meiotic maturation in the diabetic mouse.

Although there is considerable evidence that diabetes can adversely affect meiosis in mammalian oocytes, acetylation status of oocytes in a diabetic environment remains unclear. The objective was to determine acetylation or deacetylation patterns (based on immunostaining) of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16 sites at various stages during meiosis in murine oocytes from control and diabetic mice. According to quantitative real time polymerase chain reaction (qPCR), mean ± SEM relative expression of Gcn5 (1.70 ± 0.14 at metaphase [M]I and 1.27 ± 0.01 at MII, respectively), Ep300 (1.74 ± 0.04 at MI and 1.80 ± 0.001 at MII), and Pcaf (2.01 ± 0.03 at MI and 1.41 ± 0.18 at MII) mRNA in oocytes from diabetic mice were higher than those from controls (P < 0.05), whereas there was no difference (P > 0.05) during the germinal vesicle (GV) stage between the two groups (1.23 ± 0.04 for Gcn5, 0.82 ± 0.06 for Ep300, and 0.80 ± 0.07 for Pcaf). Conversely, relative mRNA expression concentrations of Hdac1, Hdac2, Hdac3, Sirt1 and Sirt2 during the germinal vesicle stage were lower in oocytes of diabetic mice (0.24 ± 0.03 for Hdac1, 0.11 ± 0.001 for Hdac2, 0.31 ± 0.03 for Hdac3, 0.28 ± 0.02 for Sirt1, and 0.55 ± 0.02 for Sirt2; P < 0.05). Similarly, the expression concentrations of these genes at the MI stage were lower in oocytes from diabetic mice (0.79 ± 0.12 for Hdac1, 0.72 ± 0.001 for Hdac2, 0.02 ± 0.001 for Sirt1, and 0.84 ± 0.08 for Sirt2; P < 0.05). Their expression concentrations at the MII stage were also lower in oocytes from diabetic mice (0.46 ± 0.03 for Hdac1, 0.93 ± 0.01 for Hdac2, 0.56 ± 0.01 for Hdac3, 0.01 ± 0.002 for Sirt1, and 0.84 ± 0.04 for Sirt2; P < 0.05). At the MI stage, however, there was no difference in the expression of Hdac3 between the two groups of oocytes (0.96 ± 0.03; P > 0.05). Taken together, diabetes altered the intracellular histone modification system, which may have contributed to changes in histone acetylation, and may be involved in the compromised maturation rate of oocytes in diabetic humans.

Prevention of acute and chronic allograft rejection by combinations of tolerogenic dendritic cells.

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DC) helps to reduce acute allograft rejection. However, this method cannot effectively prevent grafts from infiltration of inflammatory cells and fibrosis, and thus has minimal effect on chronic allograft rejection. In this study, we used mitomycin C (MMC) to generate tolerogenic DC and demonstrated that donor (Balb/c)-derived MMC-DC could induce hyporesponsiveness of recipient (C57BL/6) T cells in vitro, potentially by inducing T-cell apoptosis, decreasing IL-2 and IL-12 secretion, and increasing regulatory T-cell numbers and IL-10 secretion. Furthermore, anti-CD154 monoclonal antibody (mAb) treatment combined with donor-derived MMC-DC prolonged the survival of the allografts in vivo. The mechanisms were similar to those in vitro. Impressively, both acute and chronic rejection were prevented when donor and F1 generation (Balb/c × C57BL/6) derived MMC-DC were injected together with anti-CD154 mAb into recipients before heart allotransplantation. In summary, we showed that donor and F1-derived tolerogenic DC have a synergistic effect on induction and maintenance of T-cell regulation and the secretion of immunosuppressive cytokines. Moreover, adoptive transfer of these two types of DC could inhibit both acute and chronic transplant rejection in mice.

Combined application of blocking antibodies and MicroRNA interference in inhibiting CD44 expression.

BACKGROUND: We sought to explore the effect of CD44 targeting on the tolerance to memory cell-mediated graft rejection. METHODS: We developed a cardiac transplantation model in nude mice and administered anti-CD44 monoclonal antibodies (mAbs) to these mice. Then, we used anti-CD44 mAb and CD44-interfering microRNA (miRNA) to inhibit CD44 expression in vitro. RESULTS: The median survival time (MST) associated with multiple intraperitoneal injections was >100 days, whereas that associated with CD4(+) Tm cells blocked CD44 and that associated with a single intraperitoneal injection of anti-CD44 mAb was 11 and 10.3 days, control group was 5.5 days. The inhibition effect of the anti-CD44 mAb in 3T3 cells significantly reduced with cell proliferation. Used CD44 miRNA in 3T3 cells, the most obvious inhibition effect of mRNA appeared at 48 hours after transfection and the inhibition decreased subsequently. In combination, antibody-mediated blocking and miRNA showed some synergistic effects. CONCLUSION: The inhibition of CD44 can significantly prolong the MST in memory models. The inhibition effect of combined application showed limitations with regard to cell proliferation and duration of action, but the short-term synergistic effect of the combined approach was stronger than the effects of individual approaches.

Phosphorylase phosphatase: new horizons for an old enzyme.

Protein phosphatase-1, originally studied as phosphorylase phosphatase, is one of the major ser/thr protein phosphatases. It has a long history and a complex enzymology. It consists of a catalytic subunit of 37 kDa, which is bound to a number of different regulatory or targeting subunits. These are believed to restrict its activity to its immediate microenvironment and thus define its specificity, as well as acting to regulate phosphatase activity. The existence of multiple protein phosphatase-1 binding proteins provides the mechanism whereby phosphatase-1 activity can be involved in a diverse range of cellular functions, and reflects a novel strategy for its evolutionary development.

The effects of leflunomide and cyclosporin A on rejection of cardiac allografts in the rat.

Leflunomide is a new low molecular weight immunosuppressive drug which inhibits the enzymes dehydroorotate-dehydrogenase and protein tyrosine kinase, both of which are important components in the immune response. As the mechanisms of action of leflunomide and cyclosporin A (CsA) are different, we postulated a synergistic effect of the two drugs and tested graft survival following leflunomide administration alone or in combination with CsA in a rat cardiac transplantation model. Low- and high-responder rat strain combinations were used in parallel and the experiments were performed both with and without challenge with Linomide, an immunomodulator which promotes graft rejection in this model. In the low-responder rat strain combination (Piebald Virol Glaxo graft to Dark Agouti recipient; PVG to DA), graft survival appeared to be a dichotomous variable, being characterized by tolerance or early rejection. Leflunomide (10 or 5 mg/kg) given for 10 days induced tolerance and CsA did likewise; the addition of Linomide abolished the immunosuppressive effect of leflunomide but not that of CsA. In the high-responder combination (DA to PVG), no tolerance was seen and graft survival was moderately prolonged both after leflunomide and after CsA treatment; the addition of Linomide to CsA or to leflunomide (5 mg/kg) abolished the immunosuppressive effect of the drugs. However, when CsA-Linomide or leflunomide-Linomide were supplemented with the second immunosuppressive drug, leflunomide or CsA respectively, graft survival was significantly prolonged (P < 0.001 in both cases). This suggests leflunomide and CsA have additive potential.

The primary structure of a hemorrhagic factor, HR2b, from the venom of Okinawa habu (Trimeresurus flavoviridis).

The complete amino acid sequence of a hemorrhagic factor, HR2b, from the venom of Okinawa habu was determined. The hemorrhagic factor was fragmented by CNBr cleavage, trypsin, staphylococcal protease V8 and lysyl endopeptidase digestions. The resulting peptides were purified on high performance chromatography, and sequenced by Edman degradation. HR2b was composed of 204 amino acids with pyroglutamyl residue at the amino terminus, and the calculated mol. wt based on the amino acid composition was 23,335. There are three disulfide linkages in the primary structure. The consensus sequence (His-Glu-Xaa-Xaa-His) for zinc-binding site of zinc-requiring metalloproteinases was found in the structure. The primary structure of HR2b shows a significant similarity with that of HR2a of Amami habu venom; 98.5% identity.

Isolation of peptides homologous to domains of human alpha 1B-glycoprotein from a mongoose antihemorrhagic factor.

Thirteen peptides, homologous to one of the five domains of human alpha 1B-glycoprotein (alpha 1BG), were isolated from a mongoose antihemorrhagic factor (AHF1); four of them were generated by BrCN cleavage, and three and six peptides by the digestions with lysyl endopeptidase and staphylococcal protease V8, respectively. The purified peptides covered 75.9% of the whole sequence of human alpha 1BG (359/474 residues) and showed 46.4% identity (167/360 amino acids) with the sequence of human alpha 1BG including cysteine residues forming disulfide linkages. One of the sugar binding sites of human alpha 1BG was also conserved in AHF1. These results suggest that AHF1 is a protein homologous to human alpha 1BG and a supergene family of immunoglobulins.

Characterization of the antihemorrhagic factors of mongoose (Herpestes edwardsii).

Three antihemorrhagic factors (AHF1, AHF2 and AHF3) isolated from the serum of mongoose (Herpestes edwardsii) are glycoproteins of monomer structure with the same mol. wt (about 65,000), which contain 4.2%, 13.6% and 6.0% carbohydrates as glucose, respectively. All are composed of about 600 amino acids of similar composition. The 32 amino terminal amino acid sequences of three antihemorrhagic factors were determined, and sequence homologies were examined. AHF1 and AHF2 were of the same amino acid sequence, and showed high homologies to AHF3, oprin (opossum proteinase inhibitor) and human alpha 1B-glycoprotein; 68.7%, 42.3% and 50.0% identity, respectively. AHF1 completely inhibited the hemorrhagic activity of HR2b, the hemorrhagic factor of habu snake, at the concentration of five-fold molar excess, although incomplete inhibition (50%) of proteinase activity of the hemorrhagic factor was observed even at the concentration of 20-fold molar excess of antihemorrhagic factor. Incubation of HR2b with AHF1, and analysis of the reaction products by chromatography on TSK gel G-3000SW and on the ultracentrifuge did not show formation of an inactive enzyme inhibitor complex. However, the complex formation between AHF1 and HR2b was observed by a BIAcore analysis and TSK gel SP-5PW column chromatography. No alteration in the primary or the secondary structure of both factors was demonstrated by SDS-PAGE and circular dichroism spectrum at the far-UV wavelength before and after incubation of both factors, respectively.