The suppressive effect on CD4 T cell alloresponse against endothelial HLA-DR via PD-L1 induced by anti-A/B ligation.

While donor-specific human leukocyte antigen (HLA) antibodies are a frequent cause for chronic antibody-mediated rejection in organ transplantation, this is not the case for antibodies targeting blood group antigens, as ABO-incompatible (ABO-I) organ transplantation has been associated with a favorable graft outcome. Here, we explored the role of CD4 T cell-mediated alloresponses against endothelial HLA-D-related (DR) in the presence of anti-HLA class I or anti-A/B antibodies. CD4 T cells, notably CD45RA-memory CD4 T cells, undergo extensive proliferation in response to endothelial HLA-DR. The CD4 T cell proliferative response was enhanced in the presence of anti-HLA class I, but attenuated in the presence of anti-A/B antibodies. Microarray analysis and molecular profiling demonstrated that the expression of CD274 programmed cell death ligand 1 (PD-L1) increased in response to anti-A/B ligation-mediated extracellular signal-regulated kinase (ERK) inactivation in endothelial cells that were detected even in the presence of interferon-γ stimulation. Anti-PD-1 antibody enhanced CD4 T cell proliferation, and blocked the suppressive effect of the anti-A/B antibodies. Educated CD25+ CD127- regulatory T cells (edu.Tregs ) were more effective at preventing CD4 T cell alloresponses to endothelial cells compared with naive Treg ; anti-A/B antibodies were not involved in the Treg -mediated events. Finally, amplified expression of transcript encoding PD-L1 was observed in biopsy samples from ABO-I renal transplants when compared with those from ABO-identical/compatible transplants. Taken together, our findings identified a possible factor that might prevent graft rejection and thus contribute to a favorable outcome in ABO-I renal transplantation.

Characteristics of tissue distribution of various polysaccharides as drug carriers: influences of molecular weight and anionic charge on tumor targeting.

Using the Walker 256 model for carcinosarcoma-bearing rats, we intravenously administered 5 polysaccharide carriers with various molecular weights (MWs) and electric charges and tested for their plasma and tissue distribution. Two carriers, carboxymethylated-D-manno-D-glucan (CMMG) and CMdextran (CMDex), showed higher plasma AUC than the other carriers tested, namely, CMchitin (CMCh), N-desulfated N-acetylated heparin (DSH), and hyaluronic acid (HA). This was consistently found to be true over the range of MWs tested. For CMDex, the maximum value of plasma AUC was obtained when the MW exceeded 150 kDa. As for the anionic charge, CMDex (110-180 kDa) with a degree of substitution (DS) of the CM groups ranging from 0.2 to 0.6, showed maximum plasma AUC values. Twenty-four hours after administration, the concentration of CMDex (180-250 kDa; DS: 0.6-1.2) in tumors was more than 3% of dose/g--approximately 10-fold higher than those observed with CMCh, DSH and HA. Doxorubicin (DXR) was bound to these carriers via a peptide spacer, GlyGlyPheGly (GGFG), to give carrier-GGFG-DXR conjugates (DXR content: 4.2-7.0 (w/w)%), and the antitumor effects of these conjugates were tested with Walker 256 carcinosarcoma-bearing rats by monitoring the tumor weights after a single intravenous injection. Compared with free DXR, CMDex-GGFG-DXR and CMMG-GGFG-DXR conjugates significantly suppressed tumor growth, while the CMCh-GGFG-DXR, DSH-GGFG-DXR, and HA-GGFG-DXR conjugates in a similar comparison showed weak tumor growth inhibition. These findings suggest that the antitumor effect of the carrier-DXR conjugates was related to the extent with which the carriers accumulated in the tumors.

Improved in vivo antitumor efficacy and reduced systemic toxicity of carboxymethylpullulan-peptide-doxorubicin conjugates.

The antitumor efficacy of the conjugate of doxorubicin (DXR) and carboxymethylpullulan (CMPul) with Phe-Gly spacer (CMPul-FG-DXR) was evaluated using murine tumor models and compared with that of DXR. The conjugate exhibited higher antitumor efficacy against Lewis lung carcinoma than DXR. Complete tumor regression followed by long-term tumor-free survival was frequently observed when CMPul-FG-DXR was administered i.v. three times at a dose equivalent to 10 mg / kg of DXR. The superior survival as well as anti-metastatic effect of CMPul-FG-DXR in comparison with DXR was also demonstrated with the M5076 murine reticulosarcoma model. Body weight loss in mice treated with the conjugate was less than that in the DXR-treated group, indicating lower systemic toxicity of CMPul-FG-DXR. Simply mixing CMPul with DXR did not enhance the antitumor activity of DXR, showing that the conjugation of DXR with CMPul is necessary for improved antitumor activity. However, no enhanced antitumor efficacy of the conjugates was observed against a non-solid tumor model such as P388 leukemia. In summary, improved antitumor efficacy with reduced systemic toxicity of CMPul-FG-DXR was demonstrated in the present study. CMPul-FG-DXR may be useful as a cancer chemotherapy agent against solid tumors and metastases.

Distribution characteristics of carboxymethylpullulan-peptide-doxorubicin conjugates in tumor-bearing rats: different sequence of peptide spacers and doxorubicin contents.

Plasma and tissue distribution of conjugates of carboxymethylpullulan (CMPul) and doxorubicin (DXR), either bound directly or through three types of tetrapeptide spacers, was studied after intravenous injection to rats bearing Walker 256 carcinosarcoma and compared with that of DXR. In contrast to DXR, each conjugate retained high levels of DXR in the conjugated form in plasma and displayed high accumulation in the tumor at 6 h after the administration. Disposition characteristics of [3H]CMPul in rats bearing Walker 256 carcinosarcoma indicate that pullulan, which had molecular weight over 50 kDa, is a suitable macromolecular carrier for tumor targeting in cancer chemotherapy by carboxymethylation. We find that the in vivo antitumor effect of the conjugates depends on the tumor AUC of free DXR released from the conjugates. CMPul-DXR conjugates were also distributed in the reticuloendothelial organs, such as liver, spleen and bone marrow; however, the tissue concentrations of the conjugates in the heart, lung and muscle were lower than those of DXR. We next investigated the effect of the DXR contents of CMPul-DXR conjugates on their body distribution in rats bearing Walker 256. The half life of CMPul-DXR conjugates in plasma were shorter and the conjugates had greater accumulation in the reticuloendothelial system, while they showed lower concentrations in the tumor with increasing DXR contents. Antitumor activity of CMPul-DXR conjugates were reduced and the lethal toxicities of CMPul-DXR conjugates were amplified with increasing DXR contents.

Structure-activity relationships of carboxymethylpullulan-peptide-doxorubicin conjugates--systematic modification of peptide spacers.

A series of carboxymethylpullulan (CMPul)-doxorubicin (DXR) conjugates bound by peptide spacers of different compositions and lengths were prepared and evaluated for their in vivo antitumor effects. Systematic study of the peptide spacers indicated that CMPul-DXR conjugates bound via appropriate dipeptide spacers were more potent than DXR.

Sensitive detection of the binding of E2F to its promoter by exonuclease III- and BssHII-protection PCR assays.

For the non-radioactive, sensitive detection of the binding of transcription factor E2F to its binding site (E2 promoter), exonuclease III (ExoIII)- and BssHII-protection PCR assays were established. The binding of glutathione S-transferase E2F-1 (GST-E2F-1) fusion protein to its promoter protected the promoter against ExoIII- and BssHII-digestion. For the BssHII-protection PCR assay, a BssHII restriction site was made in the E2 promoter sequence by changing one base-pair next to its sequence. To detect E2F binding in ExoIII- or BssHII-protection PCR assays, the use of 3.13 fmol (5.00 ng) or 2.33 fmol (4.62 ng) of DNA (containing E2 promoters) and 0.325 microg (3.70 pmol) or 0.175 microg (2.00 pmol) of GST-E2F-1 protein, respectively, were found to be sufficient.

Antitumor effects and toxicities of carboxymethylpullulan-peptide-doxorubicin conjugates.

In vivo antitumor effects of the conjugates of doxorubicin (DXR) with carboxymethylpullulan (CMPul) through tetrapeptide spacers were compared with those of DXR against tumor-bearing rats. CMPul-DXR conjugates bound through Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly spacers were found to be more potent than DXR after a single intravenous injection in rats bearing Walker 256 carcinosarcoma. These conjugates were also more effective than DXR in rats bearing Yoshida sarcoma. However, CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly was less effective against Walker 256-bearing rats than DXR. Body weight loss of CMPul-DXR conjugates in rats, on the other hand, was less than that of DXR at a DXR dose of 10 mg/kg. Lethal doses of CMPul-DXR conjugates in CDF1 mice were about 3-times higher than that of DXR. These data suggest that the therapeutic index of CMPul-DXR conjugates bound through appropriate peptide spacers was increased more than that of DXR. However, CMPul-DXR conjugates tested were all less effective than DXR against Walker 256 cells in vitro. Also, 125I-labeled CMPul-DXR conjugate accumulated much less in the cells than 14C-DXR.

Orotate phosphoribosyltransferase from Thermus thermophilus: overexpression in Escherichia coli, purification and characterization.

Orotate phosphoribosyltransferase (OPRTase, EC2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). To obtain heat-stable OPRTase and to elucidate the mechanism of heat stability, this enzyme from Thermus thermophilus was expressed in Escherichia coli and purified. The pyrE gene of T. thermophilus which encodes OPRTase, contains an open reading frame of 549 base pairs with 69% G+C content. Since this gene expressed itself inefficiently in E. coli, the 5' and 3' ends of the coding regions were replaced with synonymous codons which contain more A+T and corresponds to major codons for E. coli. Introduction of the modified gene fragments into a plasmid having a tac promoter resulted in production of a polypeptide of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) in E. coli. This protein represented as much as 16% of the bacterial total protein and showed the OPRTase activity. Three purification steps, consisting of heat treatment at 65 degrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypeptide. The optimum activity of the purified OPRTase was observed at 150 mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 microM PRPP. The activation energy of this enzyme reaction was 20.3 kJ/mol. The Km of this enzyme for orotate as a substrate was 75 microM and the maximum specific activity was 300 units/mg protein under the optimum conditions. The purified OPRTase was stable for 20 min at 85 degrees C.

Synthesis of carboxymethylpullulan-peptide-doxorubicin conjugates and their properties.

The amino group of doxorubicin (DXR) was found to be bound to the carboxyl group of carboxymethylpullulan (CMPul) either directly or through tetrapeptide spacers, including Gly-Gly-Phe-Gly, Gly-Phe-Gly-Gly and Gly-Gly-Gly-Gly. These conjugates had DXR contents of 6.1-7.1%, with the degree of substitution of carboxymethyl groups being 0.6 per sugar moiety. These conjugates associate in phosphate-buffered saline (PBS) (pH 7.4), forming micelles with hydrophobic DXR inside and hydrophilic CMPul on the outside. The amounts of DXR released from the conjugates in the presence of rat liver lysosomal enzymes were determined by HPLC. The rate of the drug release differed among the conjugates tested. CMPul-DXR conjugate bound through Gly-Gly-Phe-Gly released 35% of its DXR over 24 h. On the other hand, CMPul-DXR conjugate without spacer released no free DXR. The antitumor effect of each conjugate in rats bearing Walker 256 was studied by monitoring the tumor weights after a single intravenous injection. Compared with DXR, CMPul-DXR conjugates bound through Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly spacers significantly suppressed the tumor growth, while CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly showed less antitumor effect than DXR. CMPul-DXR conjugate without spacer showed no in vivo antitumor effect even at a dose equivalent to as much as 20 mg/kg of DXR.