The Leuven Immunomodulatory Protocol Promotes T-Regulatory Cells and Substantially Prolongs Survival After First Intestinal Transplantation.

Intestinal transplantation (ITx) remains challenged by frequent/severe rejections and immunosuppression-related complications (infections/malignancies/drug toxicity). We developed the Leuven Immunomodulatory Protocol (LIP) in the lab and translated it to the clinics. LIP consists of experimentally proven maneuvers, destined to promote T-regulatory (Tregs)-dependent graft-protective mechanisms: donor-specific blood transfusion (DSBT); avoiding high-dose steroids/calcineurin-inhibitors; and minimizing reperfusion injury and endotoxin translocation. LIP was tested in 13 consecutive ITx from deceased donors (2000-2014) (observational cohort study). Recipient age was 37 years (2.8-57 years). Five-year graft/patient survival was 92%. One patient died at 9 months due to aspergillosis, another at 12 years due to nonsteroidal anti-inflammatory drug-induced enteropathy. Early acute rejection (AR) developed in two (15%); late AR in three (23%); all were reversible. No chronic rejection (CR) occurred. No malignancies developed and estimated glomerular filtration rate remained stable post-Tx. At last follow-up (3.5 years [0.5-12.5 years]), no donor-specific antibodies were detected and 11 survivors were total parenteral nutrition free with a Karnofsky score >90% in 8 recipients (follow-up >1 years). A high frequency of circulating CD4+ CD45RA- Foxp3hi memory Tregs was found (1.8% [1.39-2.21]), comparable to tolerant kidney transplant (KTx) recipients and superior to stable immunosuppression (IS)-KTx, KTx with CR, and healthy volunteers. In this ITx cohort we show that DSBT in a low-inflammatory/pro-regulatory environment activates Tregs at levels similar to tolerant-KTx, without causing sensitization. LIP limits rejection under reduced IS and thereby prolongs long-term survival to an extent not previously attained after ITx.

2016 Comprehensive Update of the Banff Working Group on Liver Allograft Pathology: Introduction of Antibody-Mediated Rejection.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Intestinal transplantation: from the laboratory to the clinics.

The intestine has long been seen as a "forbidden" organ to transplant. This is because the first attempts at Intestinal Transplantation (ITx) were defeated by rejection, technical problems, infection and graft versus host disease. Results of ITx have improved in the short-term (70 to 80% 1-year patient survival) but remain inferior to other solid organ transplants in the long-term (5 years patient survival of 50% or less). Reasons for this difference between intestine and other organ transplants are reviewed. Development of immunomodulatory protocols--e.g. protocols aiming at reducing the rejection response and facilitating engraftment--are described. Our center experience with a consecutive series of five intestinal transplants utilizing a new protolerogenic protocol and low immunosuppression is described. At time of writing, these five patients are rejection-free, nutritionally independent and lead a normal life.

Nitric oxide synthase is induced in tumor promoter-sensitive, but not tumor promoter-resistant, JB6 mouse epidermal cells cocultured with interferon-gamma-stimulated RAW 264.7 cells: the role of tumor necrosis factor-alpha.

Expression of inducible nitric oxide synthase (iNOS) has been reported to be involved in certain organs of potential tumorigenesis, including the stomach and colon. The mechanisms for iNOS expression in epithelial cells, however, are not fully understood. In the present study, we investigated the role of macrophages in epithelial iNOS expression by coculturing a stimulated murine macrophage-like cell line, RAW 264.7, with either tumor promoter-sensitive (P+) or promoter-resistant (P-) JB6 murine epidermal cells. After monoculture, treatment of RAW 264.7 cells with IFN-gamma for 24 h generated a large amount of nitrite (NO2-), as reported previously, whereas no increase in NO2- concentration was observed in the IFN-gamma-treated P+ or P-subclones. Interestingly, when IFN-gamma-treated RAW 264.7 cells were cocultured with P+ but not P- cells, we observed a marked increase in NO2- concentration (30.8+/-3.6 microM), which significantly exceeded (P < 0.01) the sum of the concentrations (20.0+/-2.3 microM) added from each cell line monoculture. Western blotting analysis revealed that, after coculture, iNOS protein was up-regulated 55-fold more than the control in JB6 P+ but not in P- cells. IFN-gamma-treated RAW 264.7 cells secreted proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. The addition of IFN-gamma-treated RAW 264.7 cell-conditioned media to P+ subclones led to a significant enhancement of NO2- formation that was diminished by the TNF-alpha-specific but not IL-1beta-specific antibody. When combined with IFN-gamma, the recombinant TNF-alpha (1-100 ng/ml) enhanced NO2- formation in JB6 P+ cells, whereas IL-1beta (1-100 ng/ml) did not. These results led us to conclude that IFN-gamma-treated RAW 264.7 cells release TNF-alpha to induce iNOS expression in promoter-sensitive JB6 cells. Thus, we propose the hypothesis that macrophages stimulate neoplastic cells with TNF-alpha via a paracrine loop to induce epithelial iNOS protein expression.

Inhibitory effect of citrus nobiletin on phorbol ester-induced skin inflammation, oxidative stress, and tumor promotion in mice.

The intake of citrus fruits has been suggested as a way to prevent the development of some types of human cancer. Nitric oxide (NO) is closely associated with the processes of epithelial carcinogenesis. We attempted a search for NO generation inhibitors in Citrus unshiu. The active constituent was traced by an activity-guiding separation. NO and superoxide (O2-) generation was induced by a combination of lipopolysaccharide and IFN-gamma in mouse macrophage RAW 264.7 cells, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocyte HL-60, respectively. Expression of inducible NO synthase and cyclooxygenase 2 proteins were detected by Western blotting. The in vivo anti-inflammatory and antitumor promoting activities were evaluated by topical TPA application to ICR mouse skin with measurement of edema formation, epidermal thickness, leukocyte infiltration, hydrogen peroxide production, and the rate of proliferating cell nuclear antigen-stained cells. As a result, nobiletin, a polymethoxyflavonoid, was identified as an inhibitor of both NO and O2- generation. Nobiletin significantly inhibited two distinct stages of skin inflammation induced by double TPA application [first stage priming (leukocyte infiltration) and second stage activation (oxidative insult by leukocytes)] by decreasing the inflammatory parameters. It also suppressed the expression of cyclooxygenase-2 and inducible NO synthase proteins and prostaglandin E2 release. Nobiletin inhibited dimethylbenz[a]anthracene (0.19 micromol)/TPA (1.6 nmol)-induced skin tumor formation at doses of 160 and 320 nmol by reducing the number of tumors per mouse by 61.2% (P < 0.001) and 75.7% (P < 0.001), respectively. The present study suggests that nobiletin is a functionally novel and possible chemopreventive agent in inflammation-associated tumorigenesis.

Purification of two forms of the associated 3-dehydroquinate hydro-lyase and shikimate:NADP+ oxidoreductase in Phaseolus mungo seedlings.

Two associated enzymes, 3-dehydroquinate hydro-lyase (EC 4.2.1.10) and shikimate:NADP+ oxidoreductase (EC 1.1.1.25), have been purified from Phaseolus mungo seedlings. These enzymes were purified 6900- and 9700-fold, respectively, but they were not separable. Moreover, two activity bands of the shikimate:NADP+ oxidoreductase were detected after polyacrylamide gel electrophoresis and the two peaks also have 3-dehydroquinate hydro-lyase activity. The two forms of the associated enzymes showed only small differences in molecular weight, Km value, pH optimum and the responses to some inhibitors.

Molecular cloning and expression patterns of three putative functional aldehyde oxidase genes and isolation of two aldehyde oxidase pseudogenes in tomato.

The final steps in the biosynthesis of the plant hormones abscisic acid (ABA) and indole-3-acetic acid (IAA) have been shown to be catalyzed by aldehyde oxidases (AO). We have cloned three putative functional AO genes (TAO1, TAO2 and TAO3) and two putative AO pseudogenes (TAO4 and TAO5) in tomato. The TAO1 cDNA described here includes the correct amino terminus of the encoded TAO1 protein and is different at the 5'-end from the TAO1 sequence in GenBank (accession number U82558). Northern analysis shows that TAO1 is expressed mainly in vegetative tissues and TAO2 is expressed in both vegetative and reproductive tissues. TAO3 expression was not detectable by Northern hybridization. These results suggest that each AO may play different roles in the regulation of tomato growth and development.

Change of choline metabolism in rat liver on chronic ethionine-feeding.

On chronic ethionine-feeding (0.1% DL-ethionine in 0.5% sucrose solution) for 2, 4 or 6 months, choline metabolism in rat hepatic cells was altered considerably, although RNA contents were virtually unchanged. Choline dehydrogenase activity in the hepatic mitochondrial fraction was suppressed by about 1/2 or 1/3, compared to its normal level, whereas choline kinase activity, which existed in the cytoplasmic fraction, was elevated more than 1.5-fold. The normal value for choline-metabolizing enzymes in terms of the choline dehydrogenase/choline kinase activity ratio was estimated to be about 70 under the defined conditions, while the average value of the activity ratio drastically changed to about 10-20 on chronic ethionine-feeding. The present results suggest that an alteration of hepatic choline-flux occurred, due both to an increase in choline kinase activity and to a counteractive decrease in choline dehydrogenase activity.

Transcriptional regulation of PS-IAA4/5 and PS-IAA6 early gene expression by indoleacetic acid and protein synthesis inhibitors in pea (Pisum sativum).

The transcription of two genes, PS-IAA4/5 and PS-IAA6, in pea is induced by indoleacetic acid (IAA) and protein synthesis inhibitors such as cycloheximide (CHX) and anisomycin (ANI). Induction by IAA is rapid, taking 5 and 7.5 minutes for PS-IAA4/5 and PS-IAA6, respectively, and is independent of IAA concentration and whether IAA has a free or esterified carboxyl group (ethyl-IAA). The rate of mRNA accumulation, however, is dependent on hormone concentration, and is greater with IAA than with ethyl-IAA. The turnover rates (t1/2) of the PS-IAA4/5 and PS-IAA6 mRNAs are 60 and 75 minutes, respectively, and are not affected by IAA. CHX or ANI induce the transcription of PS-IAA4/5 and PS-IAA6 more slowly than IAA (5 to 10 minutes for PS-IAA4/5 and 20 minutes for PS-IAA6). While protein synthesis inhibitors stabilize both mRNAs, the rapidity of induction by CHX and ANI cannot be accounted for solely by mRNA stabilization. The relationship between mRNA induction and protein synthesis inhibition does not obey Michaelis-Menten kinetics, but rather is best described by a hyperbolic curve, suggesting the release of transcriptional repression by the inhibition of protein synthesis. RNA expression experiments with transgenic tobacco seedlings or with transfected pea protoplasts using PS-IAA4/5 promoter GUS or CAT fusions reveal that CHX transcriptionally activates PS-IAA4/5 gene expression. Thus, protein synthesis inhibitors have a dual effect on PS-IAA4/5 and PS-IAA6. (1) They stabilize both mRNAs (possibly by a translational arrest-linked process or by preventing the synthesis of a labile nuclease(s)). (2) They activate transcription (possibly by preventing the synthesis or function of a repressor).

An in Vitro System of Indole-3-Acetic Acid Formation from Tryptophan in Maize (Zea mays) Coleoptile Extracts.

The formation of a product from tryptophan that had the same retention time as that of authentic indole-3-acetic acid (IAA) on high performance liquid chromatography was detected in crude extracts of maize (Zea mays) coleoptiles. The product was identified as IAA by mass spectrometry. The IAA-forming activity was co-purified with an indole-3-acetaldehyde (IAAld) oxidase activity by chromatography on hydrophobic and gel filtration (GPC-100) columns. During purification, the IAA-forming activity, rather than that of IAAld oxidase, decreased; but when hemoprotein obtained from the same tissue was added, activity recovered to the same level as that of IAAld oxidase. The promotive activity of the hemoprotein was confirmed by the result that the activity coincided with amounts of the hemoprotein after GPC-100 column chromatography. The hemoprotein was characterized and identified as a cytosolic ascorbate peroxidase (T. Koshiba [1993] Plant Cell Physiol [in press]). The reaction of the IAA-forming activity was apparently one step from tryptophan. The activity was inhibited by 2-mercaptoethanol. The optimum temperature for the IAA-forming system as well as for the IAAld oxidase was 50 to 60[deg]C, and the acitivity at 30[deg]C was one-third to one-half of that at 60[deg]C. The system did not discriminate the L- and D-enantiomers of tryptophan.

Purification and Properties of Flavin- and Molybdenum-Containing Aldehyde Oxidase from Coleoptiles of Maize.

Aldehyde oxidase (AO; EC 1.2.3.1) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L.) seedlings. The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of 150-kD subunits. It contained flavin adenine dinucleotide, iron, and molybdenum as prosthetic groups and had absorption peaks in the visible region (300-600 nm). To our knowledge, this is the first demonstration of the presence of flavin adenine dinucleotide and metals in plant AO. Other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde also served as good substrates, but N-methylnicotinamide, a good substrate for animal AO, was not oxidized. 2-Mercaptoethanol, p-chloromercu-ribenzoate, and iodoacetate partially inhibited the activity, but well-known inhibitors of animal AO, such as menadione and estradiol, caused no reduction in activity. These results indicate that, although maize AO is similar to animal enzymes in molecular mass and cofactor components, it differs in substrate specificity and susceptibility to inhibitors. Immunoblotting analysis with mouse polyclonal antibodies raised against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptiles. The possible role of this enzyme is discussed in relation to phytohormone biosynthesis in plants.

Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression.

To examine the expression of the stromal cell-derived factor 1 (SDF-1)/CXCR4 receptor ligand system in pancreatic cancer cells and endothelial cells, we performed immunohistochemical analysis for 52 pancreatic cancer tissue samples with anti-CXCR4 antibody and reverse transcription-PCR analysis for CXCR4 and SDF-1 in five pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, and PANC-1), an endothelial cell line (HUVEC), and eight pancreatic cancer tissues. We then performed cell migration assay on AsPC-1 cells, HUVECs, and CFPAC-1 cells in the presence of SDF-1 or MRC-9 fibroblast cells. Immunoreactive CXCR4 was found mainly in pancreatic cancer cells and endothelial cells of relatively large vessels around a tumorous lesion. The immunopositive ratio in the pancreatic cancer was 71.2%. There was no statistically significant correlation with clinicopathological features. SDF-1 mRNA expressions were detected in all pancreatic cancer tissues but not in pancreatic cancer cell lines and HUVECs; meanwhile, CXCR4 mRNA was detected in all pancreatic cancer tissues, cancer cell lines, and HUVECs. The results indicate that the paracrine mechanism is involved in the SDF-1/CXCR4 receptor ligand system in pancreatic cancer. In vitro studies demonstrated that SDF-1 significantly increased the migration ability of AsPC-1 and HUVECs, and these effects were inhibited by CXCR4 antagonist T22, and that the coculture system with MRC-9 also increased the migration ability of CFPAC-1 cells, and this effect was significantly inhibited by T22. Our results suggested that the SDF-1/CXCR4 receptor ligand system may have a possible role in the pancreatic cancer progression through tumor cell migration and angiogenesis.

The roles of CD25+CD4+ regulatory T cells in operational tolerance after living donor liver transplantation.

UNLABELLED: Recent evidence suggests that CD4+CD25+ regulatory T cells (Tregs) affect immune responses, including those to alloantigens in organ transplants. We have followed a group of liver allograft recipients with good liver graft function who have been weaned off immunosuppression (IS). The purpose of this study was to determine whether Tregs contributed functionally to the mechanisms of graft acceptance. MATERIAL AND METHODS: The functional assay used peripheral blood obtained from LTx recipients free of immunosuppression. The Whole population of CD4+ T cells and the CD4+ T cells depleted of CD4+CD25 high cells were tested for proliferation against donor versus third party stimulators. Moreover to determine the antigen-specificity of the Tregs, serially diluted numbering of CD4+CD25+ T cells were co-cultured with CD4+CD25- T cells. The proliferation responses were examined toward donor versus third party stimulators. RESULT: CD4+ T cells from all LTx recipients off immunosuppression showed hyporesponsiveness to the donor but not to third party stimulators. However, even after depletion of the CD4+CD25 high population, the cells continued to be hyporesponsive toward the donor. In four out of five cases, the suppression exhibited by CD4+CD25+ T cells was more specific for the donor. DISCUSSION: These findings suggest that donor alloantigen specific regulation by Tregs is one of multiple mechanisms that may contribute to the maintenance of liver graft survival in the absence of immunosuppression.

Proinflammatory and antiinflammatory cytokine production during ischemia-reperfusion injury in a case of identical twin living donor liver transplantation using no immunosuppression.

BACKGROUND: Bolus steroids are usually administered prior to graft reperfusion in an attempt to provide protection against ischemia reperfusion injury (IRI). However, the anti-IRI properties of steroids have not been established. Living donor liver transplantation (LDLT) between identical twins provides a unique opportunity to study the natural production of cytokines during transplantation without the confounding influences of the alloimmune response or of immunosuppression in particular steroids. METHODS: A 38-year-old male with hepatitis C virus-related cirrhosis and multiple hepatocellular carcinomas received a hepatic right lobe graft from his identical twin. No immunosuppression was administered, not even intraoperative bolus steroids. IRI was assessed by serum transaminases as well as by proinflammatory interleukin (IL) IL-1beta, tumor necrosis factor (TNF)-alpha, IL-8 cytokines and for potent regenerative/anti-inflammatory (IL-6, IL-10) mediators. RESULTS: Despite no administration of steroids, low peak levels of serum transaminases were observed. Serum IL-6 and IL-10 dramatically and rapidly increased during liver transplantation, namely, 160 and 20 times higher than baseline, respectively. In contrast, IL-1beta and TNF-alpha remained low during and after transplantation and an increase in IL-8 was less obvious. CONCLUSION: Syngeneic LDLT without intraoperative bolus steroids is feasible, yielding no penalty in terms of IRI. A predominance of protective cytokines was observed in the absence of steroids. Thus, the concept that intraoperative administration of steroids is necessary to protect liver transplants from IRI must be revisited.

Expression of cDNA for a bark lectin of Robinia in transgenic tobacco plants.

A cDNA encoding a bark lectin of Robinia pseudoacacia was introduced into tobacco plants. The expression of the lectin cDNA under control of the 35S promoter was confirmed by Western blot analysis and a hemagglutination assay of extracts of transgenic plants. Western blot analysis revealed that the subunit of the lectin from tobacco had a molecular mass of 29 kDa. The sequence of nine amino acids from the N-terminus of the lectin from transgenic tobacco plants was identical to that of the bark lectin from Robinia, indicating that the lectin had been processed correctly at its N-terminus in tobacco. The molecular mass of the purified native lectin produced by tobacco plants was estimated to be 112 kDa by gel filtration on a column of Superdex 200. It is suggested that the lectin subunits assembled to form tetramers in transgenic tobacco plants.

Expression of two angiogenic factors, vascular endothelial growth factor and platelet-derived endothelial cell growth factor in human pancreatic cancer, and its relationship to angiogenesis.

Tumour angiogenesis, as assayed by microvessel density (MVD), and the expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) have become established as important prognostic indicators for many tumour types. In this study, MVD and the expression of VEGF and PD-ECGF were examined by immunohistochemical staining of 50 pancreatic cancer tissues, and the relationships between either MVD or the expression of these two angiogenic factors and the clinicopathological features, including survival, were analysed. The expression of VEGF and PD-ECGF proteins were confirmed by Western blot analysis and VEGF mRNA isoforms were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in five pancreatic cancer cell lines. Twenty-eight (56%) of 50 pancreatic cancers were positive for VEGF protein in cancer cells, and 16 (32%) showed strong PD-ECGF staining in cancer and infiltrating cells. VEGF121 and VEGF165 were identified as the predominant species produced in pancreatic cancer cells. The overexpression of VEGF and PD-ECGF protein significantly correlated with high MVD (P = 0.002, 0.044, respectively). Advanced stage of disease was significantly more frequent in patients with high MVD (P = 0.025). No significant association was found between the expression of VEGF or PD-ECGF and clinicopathological features, except for tumour histology. The expression of PD-ECGF correlated with poor survival (P = 0.011), but MVD and VEGF expression were not found to be useful for the prediction of overall survival. This study suggests that VEGF and PD-ECGF may play an important role in tumour angiogenesis, and that PD-ECGF expression seems to be useful for establishing prognoses for pancreatic cancer.

Antiproliferative activity induced by the somatostatin analogue, TT-232, in human pancreatic cancer cells.

Somatostatin analogues have been developed as antiproliferative agents, but their administration as general antitumour agents is limited, mainly because of the wide distribution of somatostatin receptors throughout the human body. TT-232, a new somatostatin structural analogue, was reported to have tumour-selective antiproliferative activity without an antisecretory action. We examined whether TT-232 had antiproliferative activity in human pancreatic cancer cell lines, and compared its antiproliferative activity with that of RC-160 and other TT-232 derivatives. TT-232 inhibited the growth of all of the cell lines used in this study and induced apoptotic cell death. RC-160 showed no such growth inhibition. TT-232 also inhibited tumour formation in a xenograft model. A competitive binding assay was performed using the cell membrane fraction and 111In-DTPA-TT-232 in order to show the existence of a specific binding site on the cells. A specific binding site was detected in MIAPaCa-2 cells. It has been shown that the activation of protein tyrosine phosphatase (PTPase) is one of the main intracellular pathways responsible for somatostatinergic inhibition of cell growth. We found a significant PTPase stimulation after TT-232 administration using an immunoblot analysis assessing the level of protein tyrosine phosphorylation, and also a direct measurement of the PTPase activity. We also demonstrated that PTPase stimulation by TT-232 was involved in its antiproliferative activity as this activity was reversed by the addition of sodium orthovanadate, a PTPase inhibitor. Our results indicate that TT-232 could be a potentially useful therapeutic agent if these data are translated into clinical practice.

Idiopathic thrombocytopenic purpura after a living-related liver transplantation.

BACKGROUND: Idiopathic thrombocytopenic purpura (ITP) is a rare complication after liver transplantation. We describe three cases of ITP in pediatric patients after a living-related liver transplantation (LRLT). METHODS: Of 266 patients who underwent an LRLT between June 1990 and June 1996, severe thrombocytopenia developed in three pediatric patients after transplantation, and ITP was also diagnosed. The original disease was biliary atresia in all cases, and the patients were given a partial liver graft from a living-related mother and subsequently treated with tacrolimus and low-dose steroids as an immunosuppressive regimen. RESULTS: The duration until the onset of ITP after transplantation in the three cases was 1 day, 3 months, and 13 months, respectively. The platelet-associated IgG levels increased in all cases. A preceding viral infection was suspected in two of the three cases. All patients were treated with intravenous gamma globulin with a transient recovery of thrombocytopenia in two cases and a sustained recovery in another. CONCLUSIONS: Transplant clinicians need to be aware of the possibility of ITP complication because a sudden onset of severe thrombocytopenia can occur even in patients who are apparently doing well after undergoing an LRLT.

Inhibition of pRb phosphorylation and cell cycle progression by an antennapedia-p16(INK4A) fusion peptide in pancreatic cancer cells.

In this study, we examined whether or not a small peptide derived from p16(INK4A) protein with the antennapedia carrier sequence could inhibit the growth of pancreatic cancer cells through the inhibition of cell cycle progression. Growth inhibition by the p16-derived peptide was observed in a time- and dose-dependent manner in AsPC-1 and BxPC-3 cells (p16-negative and pRb-positive), whereas Saos-2 cells (p16-positive and pRb-negative) showed no inhibitory effect. In AsPC-1 and BxPC-3 cells, the proportion of cells in the G(1) phase markedly increased 48 h after treatment with 20 microM p16-derived peptide. Cell-cycle analysis of Saos-2 cells showed little change during the entire period of treatment. Immunoblot analysis showed inhibition of pRb phosphorylation after treatment of BxPC-3 with 10 microM p16 peptide. Furthermore, the p16 peptide caused a decrease in cyclin A at later times of treatment. These results demonstrate that the p16-derived peptide can inhibit the growth of p16-negative and pRb-positive pancreatic cancer cells by means of G(1) phase cell cycle arrest resulting from the inhibition of pRb phosphorylation. Restoration of p16/pRb tumor-suppressive pathway by re-expression of p16(INK4A) may play a therapeutic role in the treatment of pancreatic cancer.

Modifying effects of carotenoids on superoxide and nitric oxide generation from stimulated leukocytes.

Excessive and prolonged generation of superoxide (O2-) and nitric oxide (NO) from inflammatory leukocytes is associated with several lifestyle-related diseases, including cancer. In the present study, we screened 19 natural carotenoids for their modifying effects on O2- and NO generation from differentiated human promyelocytic HL-60 cells and mouse macrophage RAW 264.7 cells, respectively. Of the carotenoids tested, halocynthiaxanthin, isolated from oysters, showed the highest suppressive effect on the generation of both free radicals. The inhibitory potencies of certain carotenoids on radical generation markedly exceeded that of beta-carotene. In addition, some important structural moieties regulating radical generation suppression are discussed.