ABO incompatible renal transplantation without antibody removal using conventional immunosuppression alone.

ABO incompatible living donor renal transplantation (ABOi) can achieve outcomes comparable to ABO compatible transplantation (ABOc). However, with the exception of blood group A2 kidneys transplanted into recipients with low titer anti-A antibody, regimens generally include antibody removal, intensified immunosuppression and splenectomy or rituximab. We now report a series of 20 successful renal transplants across a range of blood group incompatibilities using conventional immunosuppression alone in recipients with low baseline anti-blood group antibody (ABGAb) titers. Incompatibilities were A1 to O (3), A1 to B (2), A2 to O (2), AB to A (2), AB to B (1), B to A1 (9), B to O (1); titers 1:1 to 1:16 by Ortho. At 36 months, patient and graft survival are 100%. Antibody-mediated rejection (AbMR) occurred in one patient with thrombophilia and low level donor-specific anti-HLA antibody. Four patients experienced cellular rejection (two subclinical), which responded to oral prednisolone. This series demonstrates that selected patients with low titer ABGAb can undergo ABOi with standard immunosuppression alone, suggesting baseline titer as a reliable predictor of AbMR. This reduces morbidity and cost of ABOi for patients with low titer ABGAb and increases the possibility of ABOi from deceased donors.

Synthetic triterpenoids, CDDO-Imidazolide and CDDO-Ethyl amide, induce chondrogenesis.

Novel methods for inducing chondrogenesis are critical for cartilage tissue engineering and regeneration. Here we show that the synthetic oleanane triterpenoids, CDDO-Imidazolide (CDDO-Im) and CDDO-Ethyl amide (CDDO-EA), at concentrations as low as 200 nM, induce chondrogenesis in organ cultures of newborn mouse calvaria. The cartilage phenotype was measured histologically with metachromatic toluidine blue staining for proteoglycans and by immunohistochemical staining for type II collagen. Furthermore, real-time polymerase chain reaction (PCR) analysis using mRNA from calvaria after 7-day treatment with CDDO-Im and CDDO-EA showed up-regulation of the chondrocyte markers SOX9 and type II collagen (alpha1). In addition, TGF-ß; BMPs 2 and 4; Smads 3, 4, 6, and 7; and TIMPs-1 and -2 were increased. In contrast, MMP-9 was strongly down-regulated. Treatment of human bone marrow-derived mesenchymal stem cells with CDDO-Im and CDDO-EA (100 nM) induced expression of SOX9, collagen IIα1, and aggrecan, as well as BMP-2 and phospho-Smad5, confirming that the above triterpenoids induce chondrogenic differentiation. This is the first report of the use of these drugs for induction of chondrogenesis.

Rotenoids mediate potent cancer chemopreventive activity through transcriptional regulation of ornithine decarboxylase.

For the discovery of new cancer chemopreventive agents, we have studied the potential of plant extracts to inhibit phorbol ester-induced ornithine decarboxylase (ODC) activity in cell culture. Four active rotenoids were obtained from the African plant Mundulea sericea (Leguminosae). These isolates were highly potent when evaluated for inhibition of chemically induced preneoplastic lesions in mammary organ culture and inhibition of papillomas in the two-stage mouse skin model, and they appear to function by a unique mechanism at the level of ODC messenger RNA expression. Based on our findings, rotenoids can be regarded as promising new chemopreventive or anticancer agents.

The effect of coordinate system selection on wrist kinematics.

Three-dimensional motion analysis of the hand and wrist is common in in-vitro and in-vivo biomechanical research. However, all studies rely on post testing analysis, where anatomical joint coordinate systems (JCS) are created to generate clinically relevant data to describe wrist motion. The purpose of this study was to present a comparison of four JCS that have been previously described in literature. Five cadaveric upper limbs were passively cycled through a flexion-extension and radial-ulnar deviation motion pathways using a wrist motion simulator. During testing, clinical wrist angle was measured using a goniometer. Following testing, wrist angle was calculated using four previously described methods of generating wrist coordinate systems, to facilitate their comparison. For flexion-extension wrist motion, only subtle difference between JCSs were detected. When comparing the performance of each JCS to the measured wrist angle during flexion-extension wrist motion, the RMSE for all three analyzed axes were all within 6.6°. For radial-ulnar deviation wrist motion, again only subtle difference between JCSs were detected. When comparing the performance of each JCS to the measured wrist angle during radial-ulnar deviation wrist motion, the RMSE for all three analyzed axes were all within 7.1°. The results of this coordinate system comparison do not favor one JCS generation method over another, as all were found to be similar and the small differences that were found are likely not clinically significant. We support using any of the analyzed coordinate system generation methods; however, a practical advantage of using certain methods is that the required digitized points to form the coordinate systems are palpable on the skin's surface.

Correction: Musashi RNA-binding protein 2 regulates estrogen receptor 1 function in breast cancer.

The original version of this article contained error in Figure 2e. In Figure 2e, the 6th colony image of T47D cells treated with shMSI2 was inadvertently replaced with a duplicate of 7th colony image. However, the conclusions reported in the manuscript are not affected by figure replacement. The authors regret that these errors were made and apologize for the confusion and inconvenience. The correct version of this figure panel appears in the Author Correction associated with this Article.

Photonic crystal lasers in InGaAsP on a SiO(2)/Si substrates and its thermal impedance.

Two-dimensional photonic crystal defect lasers in InGaAsP membranes directly bonded to a SiO(2)/Si substrate have been demonstrated. Lasing at wavelengths near 1550 nm was obtained with incident threshold pump powers as low as 1.5 mW. Good agreement between experimental data and three-dimensional finite-difference time-domain (FDTD) simulation was achieved. The thermal impedance of this laser is also characterized.

Musashi RNA-binding protein 2 regulates estrogen receptor 1 function in breast cancer.

Musashi RNA-binding protein 2 (MSI2) has important roles in human cancer. However, the regulatory mechanisms by which MSI2 alters breast cancer pathophysiology have not been clearly identified. Here we demonstrate that MSI2 directly regulates estrogen receptor 1 (ESR1), which is a well-known therapeutic target and has been shown to reflect clinical outcomes in breast cancer. Based on gene expression data analysis, we found that MSI2 expression was highly enriched in estrogen receptor (ER)-positive breast cancer and that MSI2 expression was significantly correlated with ESR1 expression, including expression of ESR1 downstream target genes. In addition, MSI2 levels were associated with clinical outcomes. MSI2 influenced breast cancer cell growth by altering ESR1 function. MSI2 alters ESR1 by binding specific sites in ESR1 RNA and by increasing ESR1 protein stability. Taken together, our findings identified a novel regulatory mechanism of MSI2 as an upstream regulator of ESR1 and revealed the clinical relevance of the RNA-binding protein MSI2 in breast cancer.

A new ligand for the peroxisome proliferator-activated receptor-gamma (PPAR-gamma), GW7845, inhibits rat mammary carcinogenesis.

We have tested a new ligand for peroxisome proliferator-activated receptor-gamma, GW7845, as an inhibitor of experimental mammary carcinogenesis, using the classic rat model with nitrosomethylurea as carcinogen. Rats were first treated with a single dose of nitrosomethylurea (50 mg/kg body weight, i.p.). Starting 1 week later, they were fed GW7845, at either 60 or 30 mg/kg of diet, for 2 months. This agent significantly reduced tumor incidence, tumor number, and tumor weight at both doses. This is the first report of the use of a ligand for peroxisome proliferator-activated receptor-gamma to prevent experimental breast cancer.

Novel triterpenoids suppress inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages.

We have synthesized more than 80 novel triterpenoids, all derivatives of oleanolic and ursolic acid, as potential anti-inflammatory and chemopreventive agents. These triterpenoids have been tested for their ability to suppress the de novo formation of two enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2), using IFN-gamma-stimulated primary mouse macrophages or lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as assay systems. Two synthetic oleananes, 3,12-dioxoolean-1-en-28-oic acid (TP-69) and 3,11-dioxoolean-1,12-dien-28-oic acid (TP-72), were highly active inhibitors of de novo formation of both iNOS and COX-2. Both TP-69 and TP-72 blocked the increase in iNOS or COX-2 mRNA induced by IFN-gamma or LPS. In addition, TP-72 suppressed NF-KB activation in primary macrophages treated with the combination of IFN-gamma and LPS or IFN-gamma and tumor necrosis factor. The 3-alpha(axial)-epimer of ursolic acid suppressed de novo formation of COX-2, in contrast to naturally occurring 3-beta(equatorial)-ursolic acid. Inhibitory effects of TP-69 or TP-72 on iNOS formation were not blocked by the glucocorticoid receptor antagonist RU-486, indicating that these triterpenoids do not act through the glucocorticoid receptor, nor does TP-72 act as an iNOS or COX-2 enzyme inhibitor when added to RAW cells in which synthesis of these two enzymes in response to LPS has already been induced. It may be possible to develop triterpenoids as useful agents for chemoprevention of cancer or other chronic diseases with an inflammatory component.

Arzoxifene, a new selective estrogen receptor modulator for chemoprevention of experimental breast cancer.

Arzoxifene ([6-hydroxy-3-[4-[2-(1-piperidinyl)-ethoxy]phenoxy]-2-(4-methoxyphenyl)]benzo[b]thiophene) is a selective estrogen receptor modulator (SERM) that is a potent estrogen antagonist in mammary and uterine tissue while acting as an estrogen agonist to maintain bone density and lower serum cholesterol. Arzoxifene is a highly effective agent for prevention of mammary cancer induced in the rat by the carcinogen nitrosomethylurea and is significantly more potent than raloxifene in this regard. Arzoxifene is devoid of the uterotrophic effects of tamoxifen, suggesting that, in contrast to tamoxifen, it is unlikely that the clinical use of arzoxifene will increase the risk of developing endometrial carcinoma.

A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity.

The new synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a potent, multifunctional molecule. It induces monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts and enhances the neuronal differentiation of rat PC12 pheochromocytoma cells caused by nerve growth factor. CDDO inhibits proliferation of many human tumor cell lines, including those derived from estrogen receptor-positive and -negative breast carcinomas, myeloid leukemias, and several carcinomas bearing a Smad4 mutation. Furthermore, it suppresses the abilities of various inflammatory cytokines, such as IFN-gamma, interleukin-1, and tumor necrosis factor-alpha, to induce de novo formation of the enzymes inducible nitric oxide synthase (iNos) and inducible cyclooxygenase (COX-2) in mouse peritoneal macrophages, rat brain microglia, and human colon fibroblasts. CDDO will also protect rat brain hippocampal neurons from cell death induced by beta-amyloid. The above activities have been found at concentrations ranging from 10(-6) to 10(-9) M in cell culture, and these results suggest that CDDO needs further study in vivo, for either chemoprevention or chemotherapy of malignancy as well as for neuroprotection.

Prospects for prevention and treatment of cancer with selective PPARgamma modulators (SPARMs).

Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor and transcription factor that regulates the expression of many genes relevant to carcinogenesis, is now an important target for development of new drugs for the prevention and treatment of cancer. Deficient expression of PPARgamma can be a significant risk factor for carcinogenesis, although in some cases overexpression enhances carcinogenesis. Ligands for PPARgamma suppress breast carcinogenesis in experimental models and induce differentiation of human liposarcoma cells. By analogy to the selective estrogen receptor modulator (SERM) concept, it is suggested that selective PPARgamma modulators (SPARMs), designed to have desired effects on specific genes and target tissues without undesirable effects on others, will be clinically important in the future for chemoprevention and chemotherapy of cancer.

Synthetic triterpenoids activate a pathway for apoptosis in AML cells involving downregulation of FLIP and sensitization to TRAIL.

Acute myelogenous leukemia (AML) remains a deadly disease for most adult patients, due primarily to the emergence of chemoresistant cells. Defects in apoptosis pathways make important contributions to chemoresistance, suggesting a need to restore apoptosis sensitivity or to identify alternative pathways for apoptosis induction. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity, including 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester (CDDO-m). We explored the effects of CDDO and CDDO-m in vitro on established AML cell lines (HL-60, U937, AML-2) and on freshly isolated AML blasts. CDDO and CDDO-m reduced the viability of all AML cell lines tested in a dose-dependent manner, with effective doses for killing 50% of cells (ED(50)) within 48 h of approximately 1 and 0.5 muM, respectively. CDDO or CDDO-m also induced substantial increases in cell death in five out of 10 samples of primary AML blasts. Cell death induced by CDDO and CDDO-m was attributed to apoptosis, based on characteristic cell morphology and evidence of caspase activation. Immunoblot analysis demonstrated proteolytic processing of caspase-3, -7, and -8, but not caspase-9, suggesting the involvement of the 'extrinsic' pathway, linked to apoptosis induction by TNF-family death receptors. Accordingly, CDDO and CDDO-m induced concentration-dependent reductions in the levels of FLIP protein, an endogenous antagonist of caspase-8, without altering the levels of several other apoptosis-relevant proteins. Reductions in FLIP were rapid, detectable within 3 h after exposure of AML cell lines to CDDO or CDDO-m. CDDO and CDDO-m also sensitized two of four leukemia lines to TRAIL, a TNF-family death ligand. The findings suggest that synthetic triterpenoids warrant further investigation in the treatment of AML, alone or in combination with TRAIL or other immune-based therapies.

A synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), is a ligand for the peroxisome proliferator-activated receptor gamma.

A novel synthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), previously reported to have potent differentiating, antiproliferative, and antiinflammatory activities, has been identified as a ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma). CDDO induces adipocytic differentiation in 3T3-L1 cells, although it is not as potent as the full agonist of PPARgamma, rosiglitazone. Binding studies of CDDO to PPARgamma using a scintillation proximity assay give a Ki between 10(-8) to 10(-7) M. In transactivation assays, CDDO is a partial agonist for PPARgamma. The methyl ester of CDDO, CDDO-Me, binds to PPARgamma with similar affinity, but is an antagonist. Like other PPARgamma ligands, CDDO synergizes with a retinoid X receptor (RXR)-specific ligand to induce 3T3-L1 differentiation, while CDDO-Me is an antagonist in this assay. The partial agonism of CDDO and the antagonism of CDDO-Me reflect the differences in their capacity to recruit or displace cofactors of transcriptional regulation; CDDO and rosiglitazone both release the nuclear receptor corepressor, NCoR, from PPARgamma, while CDDO-Me does not. The differences between CDDO and rosiglitazone as either partial or full agonists, respectively, are seen in the weaker ability of CDDO to recruit the coactivator CREB-binding protein, CBP, to PPARgamma. Our results establish the triterpenoid CDDO as a member of a new class of PPARgamma ligands.

Novel synthetic oleanane and ursane triterpenoids with various enone functionalities in ring A as inhibitors of nitric oxide production in mouse macrophages.

We initially randomly synthesized about 60 oleanane and ursane triterpenoids as potential anti-inflammatory and cancer chemopreventive agents. Preliminary screening of these derivatives for inhibition of production of nitric oxide induced by interferon-gamma in mouse macrophages revealed that 3-oxooleana-1, 12-dien-28-oic acid (B-15) showed significant activity (IC(50) = 5.6 microM). On the basis of the structure of B-15, 19 novel olean- and urs-12-ene triterpenoids with a 1-en-3-one functionality having a substituent at C-2 in ring A have been designed and synthesized. Among them, 3-oxooleana-1,12-diene derivatives with carboxyl, methoxycarbonyl, and nitrile groups at C-2 showed higher activity than the lead compound B-15. In particular, 2-carboxy-3-oxooleana-1, 12-dien-28-oic acid (3) had the highest activity (IC(50) = 0.07 microM) in this group of triterpenoids. The potency of 3 was similar to that of hydrocortisone (IC(50) = 0.01 microM), although 3 does not act through the glucocorticoid receptor. Interesting structure-activity relationships of these novel synthetic triterpenoids are also discussed.

Synthetic oleanane and ursane triterpenoids with modified rings A and C: a series of highly active inhibitors of nitric oxide production in mouse macrophages.

We have designed and synthesized 16 new olean- and urs-1-en-3-one triterpenoids with various modified rings C as potential antiinflammatory and cancer chemopreventive agents and evaluated their inhibitory activities against production of nitric oxide induced by interferon-gamma in mouse macrophages. This investigation revealed that 9(11)-en-12-one and 12-en-11-one functionalities in ring C increase the potency by about 2-10 times compared with the original 12-ene. Subsequently, we have designed and synthesized novel olean- and urs-1-en-3-one derivatives with nitrile and carboxyl groups at C-2 in ring A and with 9(11)-en-12-one and 12-en-11-one functionalities in ring C. Among them, we have found that methyl 2-cyano-3, 12-dioxooleana-1,9(11)-dien-28-oate (25), 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) (26), and methyl 2-carboxy-3,12-dioxooleana-1,9(11)-dien-28-oate (29) have extremely high potency (IC(50) = 0.1 nM level). Their potency is similar to that of dexamethasone although they do not act through the glucocorticoid receptor. Overall, the combination of modified rings A and C increases the potency by about 10 000 times compared with the lead compound, 3-oxooleana-1,12-dien-28-oic acid (8) (IC(50) = 1 microM level). The selected oleanane triterpenoid, CDDO (26), was found to be a potent, multifunctional agent in various in vitro assays and to show antiinflammatory activity against thioglycollate-interferon-gamma-induced mouse peritonitis.

Novel approach to fabricate porous sponges of poly(D,L-lactic-co-glycolic acid) without the use of organic solvents.

A novel method was developed to produce highly porous sponges for potential use in tissue engineering, without the use of organic solvents. Highly porous sponges of biodegradable polymers are frequently utilized in tissue engineering both to transplant cells or growth factors, and to serve as a template for tissue regeneration. The processes utilized to fabricate sponges typically use organic solvents, but organic residues remaining in the sponges may be harmful to adherent cells, protein growth factors or nearby tissues. This report describes a technique to fabricate macroporous sponges from synthetic biodegradable polymers using high pressure carbon dioxide processing at room temperature. Solid discs of poly (D,L-lactic-co-glycolic acid) were saturated with CO2 by exposure to high pressure CO2 gas (5.5 MPa) for 72 h at room temperature. The solubility of the gas in the polymer was then rapidly decreased by reducing the CO2 gas pressure to atmospheric levels. This created a thermodynamic instability for the CO2 dissolved in the polymer discs, and resulted in the nucleation and growth of gas cells within the polymer matrix. Polymer sponges with large pores (approximately 100 microns) and porosities of up to 93% could be fabricated with this technique. The porosity of the sponges could be controlled by the perform production technique, and mixing crystalline and amorphous polymers. Fibre-reinforced foams could also be produced by placing polymer fibres within the polymer matrix before CO2 gas processing.

A lignan and four terpenoids from Brucea javanica that induce differentiation with cultured HL-60 promyelocytic leukemia cells.

A novel lignan, guaiacylglycerol-beta-O-6'-(2-methoxy)cinnamyl alcohol either, three known simaroubolides, brusatol, dehydrobrusatol, yadanziolide C, and the known terpenoid, blumenol A, were obtained as active compounds from an ethyl acetate-soluble extract of Brucea javanica, using a bioassay based on the induction of cell differentiation with human promyelocytic leukemia (HL-60) cells. Also obtained were the known coumarinolignan, cleomiscosin A, and the known quassinoid glycoside, bruceoside B, which were inactive in the HL-60 cell test system. The structure of the new lignan was determined by a combination of 1D and 2D NMR techniques.

Discovery of natural product chemopreventive agents utilizing HL-60 cell differentiation as a model.

Terminal differentiation of human promyelocytic leukemia (HL-60) cells can be induced by a variety of chemical agents and this process can be monitored readily by the generation of morphologically, histochemically, and functionally mature granulocytes and monocytes/macrophages. The availability of this model has heightened interest in the possible therapeutic role of inducers of myeloid differentiation for the treatment of leukemia and other neoplasms. In addition, however, potent cancer chemopreventive agents induce HL-60 cell differentiation at very low dose levels. Thus, as part of our search for natural product chemopreventive agents, extracts derived from nearly 400 plants were tested for their potential to induce HL-60 cell differentiation. As a result, 17 plant extracts were judged to be active (ED50 values < or = 4 micrograms/ml). One of most potent leads was an extract derived from Dirca occidentalis Gray (Thymelaeaceae) (ED50, 0.14 micrograms/ml), and bioassay-guided fractionation led to the identification of genkwanin (I), (+/-)-lariciresinol (II) and sitoindoside II (IV) as active principles, with ED50 values of 18.3, 1.1 and 0.069 microM, respectively. Based on these data, we conclude that the HL-60 cell differentiation system is a valid and useful model for the discovery of natural product cancer chemopreventive or chemotherapeutic agents.