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.

Evaluation of carboxymethylpullulan as a novel carrier for targeting immune tissues.

PURPOSE: To demonstrate the potential of carboxymethylpullulan (CMPul) as a carrier for targeting immune tissues, and to find whether immune tissues could be set as the target of an immunosuppressant to treat autoimmune diseases. METHODS: The biodistribution of CMPul was investigated to evaluate its potency as a carrier for targeting immune tissues. Furthermore, an immunosuppressant-CMPul conjugate was prepared and its suppressive effect on rat adjuvant arthritis was examined. RESULTS: The disappearance rate of 3H-labeled CMPul from the blood circulation was much slower than that of 3H-labeled pullulan (Pul) after intravenous injection to normal rats. The concentration of 3H-labeled CMPul in the spleen and lymph nodes was much higher than that of 3H-labeled Pul at 24 hours after the injection, whereas the concentration of 3H-labeled CMPul in the liver was significantly lower than that of 3H-labeled Pul. A similar targeting property of 3H-labeled CMPul for these immune tissues was observed in arthritic rats. A conjugate composed of a novel immunosuppressant PA-48153C and CMPul showed a suppressive effect on rat adjuvant arthritis judging from a reduction of the arthritic index and spleen weight and an increase of body weight. CONCLUSIONS: CMPul is expected to be a promising carrier for targeting immune tissues with an immunosuppressant to enable treatment of autoimmune diseases.

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.

Synthesis of sialyl Lewis X-polysaccharide conjugates.

Sialyl Lewis X (SLeX) is well known as a ligand of the cell adhesion molecule E-selectin which is specifically expressed at inflammatory lesion sites. We have synthesized several SLeX-polysaccharide conjugates and examined their potential for drug delivery to inflammatory lesions. The AUC (area under the blood concentration-time curve) 0-24 h of SLeX-CMCht (1), SLeX-CMPul (2) and SLeX-DSH (3) at the inflammatory lesion was about 60-, 300-, and 30-fold higher than that of the monovalent SLeX (7), respectively. Moreover, 1 showed 2-fold higher accumulation in the inflammatory lesion than SLN-CMCht (4), and 2 showed 2.5-fold higher accumulation than SLN-CMPul (5).

Effect of the sialyl Lewis X (SLe(x)) moiety on splenic accumulation of SLe(x)-carboxymethylpullulan conjugate.

Sialyl Lewis X (SLe(x)), an E-selectin ligand, was conjugated with carboxymethylpullulan (CMPul) and the disposition characteristics of this conjugate after intravenous administration were investigated using mice with ear edema. The concentration of 3H-labeled SLe(x)-CMPul in the spleen was significantly high. When CMPul was modified with a saccharide unable to bind to E-selectin, this splenic accumulation was not observed. The uptake of radiolabeled SLe(x)-CMPul by the spleen was completely inhibited by a 100-fold molar of cold SLe(x)-CMPul but not by a sialyl N-acetyllactosamine-CMPul conjugate (SLN-CMPul). Microautoradiography analyses revealed that SLe(x)-CMPul accumulated in the marginal zone of the spleen.

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.

Syntheses and evaluation of biantennary oligosaccharide ligands mimicking sialyl Lewis X.

Sialyl Lewis X (1) is known to be a ligand of the cell adhesion molecule E-selectin. We have synthesized several biantennary glycoside-terminated ligands mimicking sialyl Lewis X (1), and evaluated their binding activity to E-selectin using HL-60 cells expressing sialyl Lewis X epitope and human umbilical vein endothelial cells (HUVECs). These compounds were found to possess moderate binding activities to E-selectin. Among them, di-fucoside analog (8) which has no sialic acid carboxylate group was more active than 2, which had both the sialyl-galactose residue and the fucose residue (IC50, 8: 4.7 mM, 2: 11.7 mM). Furthermore, in the rat pleuritic model in vivo induced by carrageenin, 8 was found to reduce neutrophil infiltration at inflammatory lesions.

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.

Enhanced accumulation of sialyl Lewis X-carboxymethylpullulan conjugate in acute inflammatory lesion.

PURPOSE: E-selectin is a cell adhesion molecule that is specifically expressed in the inflammatory vascular endothelium in response to cytokines such as IL-1 beta and TNF-alpha, and interacts with specific ligands containing sialyl Lewis X (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, SLex). In order to investigate the ability of E-selectin ligands to target the inflammatory site, the tissue distribution of carboxymethylpullulan (CMPul) modified with SLex was studied. METHODS: CMPul conjugates with various saccharides containing SLex and monovalent SLex were intravenously administered to mice with ear edema induced by arachidonic acid, and their distributions to the inflamed ear and other tissues were studied. To determine the microdistributions of these compounds, the inflamed ear was subjected to microautoradiography. RESULTS: After intravenous administration AUC0-24h of SLex-CMPul, which binds to E-selectin, in the inflamed ear was about 300-fold and 2.5-fold higher than that of monovalent SLex and CMPul conjugated with other saccharides, which can not serve as ligands for E-selectin. Microautoradiography also revealed SLex-CMPul accumulated at the microvessels in the inflammatory lesions. CONCLUSIONS: SLex-CMPul was found to have the potential to target drugs to the inflammatory lesion.

Medium temperature, 310 K, provides single crystals of orotate phosphoribosyltransferase from Thermus thermophilus.

Crystallization of orotate phosphoribosyltransferase from a thermophilic organism, Thermus thermophilus, was achieved using the hanging-drop vapour-diffusion method coupled with a macroseeding starter. Small needle-like microcrystals were grown in a fresh protein solution in the presence of 2-methyl-2,4-pentanediol at 298 K or below. Although these normal temperature conditions caused stacking crystallization, an increase of temperature to 310 K permitted crystal growth. This was because of increased enzyme solubility at the higher temperature. The crystal was found to belong to the monoclinic space group P21with unit-cell parameters a = 44.4, b = 59.6, c = 67.8 A and beta = 98.3 degrees.

Effects of C-terminal deletion on the activity and thermostability of orotate phosphoribosyltransferase from Thermus thermophilus.

To investigate the role of the C-terminal region on the activity and thermostability of orotate phosphoribosyltransferase (OPRTase, EC 2. 4.2.10) from Thermus thermophilus, four C-terminal amino acid-deleted OPRTases (1, 2, 3, and 5 residues deleted) were constructed. The activities of all the mutant OPRTases were lower than that of wild-type OPRTase at all temperatures investigated (50-80 degreesC). V- and EV-OPRTase, mutants with Val and Glu-Val deletions, respectively, showed 63 to 75% of the activity of wild-type OPRTase at the temperatures investigated. EEV- and PLEEV-OPRTase, with Glu-Glu-Val and Pro-Leu-Glu-Glu-Val deletions, respectively, had activities of 22 to 35% of the wild-type. The Km values for orotate of all mutant OPRTases were more than 4-fold higher than that of the wild-type (25 microM). On the other hand, the Km for PRPP of the wild-type was 34 microM, and there were no significant differences between the wild-type and mutant OPRTases. The kcat values of the V- and EV-OPRTases were similar to that of the wild-type, but those of the EEV- and PLEEV-OPRTases were less than 50% that of the wild-type. The optimum temperature of all mutant OPRTases, 70 degreesC, was 10 degreesC lower than that of the wild-type. The remaining activities of wild-type and V-OPRTase after incubation at 90 degreesC for 20 min were 70 and 60% of the non-treated OPRTase activity, respectively. Although the remaining activity of EV-OPRTase was only 14% of the non-treated OPRTase activity, the addition of 200 mM KCl during heat treatment increased it to 70%. Circular dichroism spectroscopy revealed that V- and EV-OPRTase denature more easily than the wild-type OPRTase. The results suggest that the C-terminal valine and glutamic acid residues are important for the activity and thermostability of T. thermophilus OPRTase.

Sialyl Lewis X-polysaccharide conjugates: targeting inflammatory lesions.

A novel system for active targeting of inflammatory lesions has been established. A SLeX-CMPul conjugate (2) showed accumulation that was 2.5-fold higher in inflammatory lesions in vivo than a SLN-CMPul conjugate (4) and 300-fold higher than monovalent SLeX (6).

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.

Hepatic accumulation of glutamic acid branched neogalactosyllipid modified liposomes.

We synthesized branched type galactosyllipid derivatives for liposome modification for the targeting of asialoglycoprotein receptors on the surface of liver cells. Galactose was coupled to the alpha- and gamma-carboxyl groups of glutamic acid via a triethyleneglycol spacer, then this glutamic moiety was bound to the lipid anchor. Ricinus communis agglutinin (RCA120) induced the agglutination of liposomes modified with mono-, bi- and tri-antennary neogalactosyllipid. With the bi- or tri-antennary derivatives, agglutination was observed at fewer galactosyl residues on the liposomes. We examined the effect of the branching structure in vivo. The difference in accumulation of liposomes between non-branched type neogalactosyllipid and branched type neogalactosyllipid was not large. Liver accumulation of liposomes depended on the galactosyl residues. The number of galactosyl residues was more effective for accumulation in the liver than for branching. We studied the effect of asialofetuin preinjection on the hepatic accumulation of neogalactosyllipid modified liposomes. Hepatic accumulation of liposomes was inhibited by preinjection of asialofetuin. The effect of preinjection was almost equal among the ligands. These results show that the saccharide density on the liposome surface seemed to be a more important factor than the branching structure of the ligand for liver targeting.

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.

Alterations in polyamine levels of nematode, earthworm, leech and planarian during regeneration, temperature and osmotic stresses.

Free-living nematodes, Caenorhabditis elegans and Dorylaimus fodori, contain putrescine and spermidine. Putrescine, spermidine and spermine occur in the parasitic Nematoda, Ascaris suum, Anisakis simplex and Dirofilaria immitis. Earthworms, Eisenia foetida, Tubifex hattai and Pheretima communissima and the leech, Hirudo nipponia (belonging to Annelida) and the planarian, Dugesia japonica (belonging to Platyhelminthes) contain homospermidine and spermine in addition to putrescine and spermidine. Regenerated heads of E. foetida and D. japonica are rich in putrescine indicating the stimulation of its synthesis during regeneration. Putrescine and spermidine levels temporarily increase after heat shock in C. elegans, E. foetida and D. japonica and cold shock and hypertonic osmotic shock treatments in D. japonica.

Occurrence of tertiary and quaternary branched polyamines in thermophilic archaebacteria.

Polyamines of thermophilic archaebacteria were analysed by high-performance liquid chromatography and gas chromatography. Thermoplasma acidophilum and Thermoplasma volcanium ubiquitously contained spermidine and spermine. Four species of Sulfolobus, S. acidocaldarius, S. solfataricus, S. metallicus and S. shibatae, two species of Acidianus, A. brierleyi and A. infernus, and Metallosphaera sedula, contained norspermidine and norspermine in addition to spermidine and spermine, but quantitative distribution profiles were species-specific. A tertiary tetra-amine, N4-aminopropylspermidine, and a quaternary penta-amine, N4-bis(aminopropyl)spermidine, were detected as a major polyamine in three species of Thermococcus, T. celer, T. litoralis and T. stetteri, and two Pyrococcus species, P. furiosus and P. woesei. This is the first report on the occurrence of branched polyamines in archaebacteria.