Androgen deprivation-induced elevated nuclear SIRT1 promotes prostate tumor cell survival by reactivation of AR signaling.

The NAD+-dependent deacetylase, Sirtuin 1 (SIRT1) is involved in prostate cancer pathogenesis. However, the actual contribution is unclear as some reports propose a protective role while others suggest it is harmful. We provide evidence for a contextual role for SIRT1 in prostate cancer. Our data show that (i) mice orthotopically implanted with SIRT1-silenced LNCaP cells produced smaller tumors; (ii) SIRT1 suppression mimicked AR inhibitory effects in hormone responsive LNCaP cells; and (iii) caused significant reduction in gene signatures associated with E2F and MYC targets in AR-null PC-3 and E2F and mTORC1 signaling in castrate-resistant ARv7 positive 22Rv1 cells. Our findings further show increased nuclear SIRT1 (nSIRT1) protein under androgen-depleted relative to androgen-replete conditions in prostate cancer cell lines. Silencing SIRT1 resulted in decreased recruitment of AR to PSA enhancer selectively under androgen-deprivation conditions. Prostate cancer outcome data show that patients with higher levels of nSIRT1 progress to advanced disease relative to patients with low nSIRT1 levels. Collectively, we demonstrate that lowering SIRT1 levels potentially provides new avenues to effectively prevent prostate cancer recurrence.

Hematopoietic knockdown of PPARδ reduces atherosclerosis in LDLR-/- mice.

PPARδ (peroxisome proliferator-activated receptor δ) mediates inflammation in response to lipid accumulation. Systemic administration of a PPARδ agonist can ameliorate atherosclerosis. Paradoxically, genetic deletion of PPARδ in hematopoietic cells led to a reduction of atherosclerosis in murine models, suggesting that downregulation of PPARδ expression in these cells may mitigate atherogenesis. To advance this finding forward to potential clinical translation through hematopoietic stem cell transplantation-based gene therapy, we employed a microRNA (miRNA) approach to knock down PPARδ expression in bone marrow cells followed by transplantation of the cells into LDLR-/- mice. We found that knockdown of PPARδ expression in the hematopoietic system caused a dramatic reduction in aortic atherosclerotic lesions. In macrophages, a key component in atherogenesis, knockdown of PPARδ led to decreased expression of multiple pro-inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1ß and IL-6. Expression of CCR2, a receptor for MCP-1, was also decreased. The downregulation of pro-inflammatory factors is consistent with significant reduction of macrophage presence in the lesions, which may also be attributable to elevation of ABCA1 (ATP-binding cassette, subfamily A, member 1) and depression of adipocyte differentiate-related protein. Furthermore, the abundance of both MCP-1 and matrix metalloproteinase-9 proteins was reduced in plaque areas. Our results demonstrate that miRNA-mediated PPARδ knockdown in hematopoietic cells is able to ameliorate atherosclerosis.

Mitochondrial genome instability resulting from SUV3 haploinsufficiency leads to tumorigenesis and shortened lifespan.

Mitochondrial dysfunction has been a hallmark of cancer. However, whether it has a causative role awaits to be elucidated. Here, using an animal model derived from inactivation of SUV3, a mitochondrial helicase, we demonstrated that mSuv3+/- mice harbored increased mitochondrial DNA (mtDNA) mutations and decreased mtDNA copy numbers, leading to tumor development in various sites and shortened lifespan. These phenotypes were transmitted maternally, indicating the etiological role of the mitochondria. Importantly, reduced SUV3 expression was observed in human breast tumor specimens compared with corresponding normal tissues in two independent cohorts. These results demonstrated for the first time that maintaining mtDNA integrity by SUV3 helicase is critical for cancer suppression.

Critical role of chemokine (C-C motif) receptor 2 (CCR2) in the KKAy + Apoe -/- mouse model of the metabolic syndrome.

AIMS/HYPOTHESIS: Chemokines and their receptors such as chemokine (C-C motif) receptor 2 (CCR2) may contribute to the pathogenesis of the metabolic syndrome via their effects on inflammatory monocytes. Increased accumulation of CCR2-driven inflammatory monocytes in epididymal fat pads is thought to favour the development of insulin resistance. Ultimately, the resulting hyperglycaemia and dyslipidaemia contribute to development of the metabolic syndrome complications such as cardiovascular disease and diabetic nephropathy. Our goal was to elucidate the role of CCR2 and inflammatory monocytes in a mouse model that resembles the human metabolic syndrome. METHODS: We generated a model of the metabolic syndrome by backcrossing KKAy ( + ) with Apoe ( -/- ) mice (KKAy ( + ) Apoe ( -/- )) and studied the role of CCR2 in this model system. RESULTS: KKAy ( + ) Apoe ( -/- ) mice were characterised by the presence of obesity, insulin resistance, dyslipidaemia and increased systemic inflammation. This model also manifested two complications of the metabolic syndrome: atherosclerosis and diabetic nephropathy. Inactivation of Ccr2 in KKAy (+) Apoe ( -/- ) mice protected against the metabolic syndrome, as well as atherosclerosis and diabetic nephropathy. This protective phenotype was associated with a reduced number of inflammatory monocytes in the liver and muscle, but not in the epididymal fat pads; circulating levels of adipokines such as leptin, resistin and adiponectin were also not reduced. Interestingly, the proportion of inflammatory monocytes in the liver, pancreas and muscle, but not in the epididymal fat pads, correlated significantly with peripheral glucose levels. CONCLUSIONS/INTERPRETATION: CCR2-driven inflammatory monocyte accumulation in the liver and muscle may be a critical pathogenic factor in the development of the metabolic syndrome.

Macrophage LXRα gene therapy ameliorates atherosclerosis as well as hypertriglyceridemia in LDLR(-/-) mice.

Liver X receptors (LXRs) are implicated in the regulation of cholesterol homeostasis, inflammatory response and atherogenesis. Administration of LXR agonists inhibits the progress of atherosclerosis, and also increases plasma triglyceride levels, representing an obstacle to their use in treating this disease. The objective of this study was to develop an alternative approach that could overcome this obstacle. Eight-week-old low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were transplanted with hematopoietic stem cell (HSC)-enriched bone marrow cells transduced with lentivectors expressing either green fluorescent protein (GFP) (Lenti-SP-GFP, control) or LXRα (Lenti-SP-LXRα) driven by a synthetic macrophage promoter. At 4 weeks post-transplant, the mice were fed with a Western diet for 8 weeks and then killed. Compared with Lenti-SP-GFP mice, the Lenti-SP-LXRα mice had a 30% reduction in atherosclerotic lesions, which was accompanied by increases in levels of macrophage expression of cholesterol efflux genes apolipoprotein E and ATP-binding cassette A1, as well as decreases in plasma inflammatory cytokines interleukin-6 and tumor necrosis factor-α. Intriguingly, a 50% reduction of plasma triglyceride level was also observed. We conclude that HSC-based macrophage LXRα gene therapy ameliorates the development of atherosclerosis along with an unexpected concomitant reduction of plasma triglyceride levels in LDLR(-/-) mice. These findings highlight the potential value of macrophage LXR expression as an avenue for therapeutic intervention against atherosclerosis.

Estrogen receptor alpha is a major mediator of 17beta-estradiol's atheroprotective effects on lesion size in Apoe-/- mice.

The inhibitory effects of estrogen (17beta-estradiol) on atherosclerosis have been well documented in numerous animal models, and epidemiological evidence supports this protective effect in humans. The detailed mechanisms for this protection are not understood, but most are thought to be mediated through estrogen receptors (ERs), of which two are known (ERalpha and ERbeta). To investigate the role of ERalpha in the atheroprotective effect of 17beta-estradiol (E2), we ovariectomized female mice that lack apoE (AAee) or lack both apoE and ERalpha (alphaalphaee), and treated half of them with E2 for three months. E2 treatment of ovariectomized AAee females dramatically reduced the size of the lesions as well as their histological complexity. Plasma cholesterol was significantly reduced in this group, although the observed extent of protection by E2 was greater than could be explained solely by the change in lipid levels. In contrast, E2 treatment of ovariectomized alphaalphaee females caused minimal reduction in lesion size and no reduction in total plasma cholesterol compared with alphaalphaee mice without E2, demonstrating that ERalpha is a major mediator of the atheroprotective effect of E2. Nevertheless, E2 treatment significantly reduced the complexity of plaques in the alphaalphaee females, although not to the same degree as in AAee females, suggesting the existence of ERalpha-independent atheroprotective effects of E2.

Preformed membrane-associated stores of interleukin (IL)-12 are a previously unrecognized source of bioactive IL-12 that is mobilized within minutes of contact with an intracellular parasite.

The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)-inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2-4 h of stimulation with lipopolysaccharide plus interferon gamma. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

Defects in the generation of IFN-gamma are overcome to control infection with Leishmania donovani in CC chemokine receptor (CCR) 5-, macrophage inflammatory protein-1 alpha-, or CCR2-deficient mice.

We investigated the immune responses in mice lacking CCR2, CCR5, or macrophage inflammatory protein-1 alpha (MIP-1 alpha), a ligand for CCR5, in two situations: following T cell stimulation or after challenge with Leishmania donovani, an intracellular microbe whose control is dependent on a Th1 immune response. Mice deficient in CCR5, MIP-1 alpha, or CCR2 had reduced IFN-gamma responses following ligation of the TCR. Reduced IFN-gamma responses following PMA and ionomycin were also observed in CD8+ T cells of CCR5-/- and CCR2-/- mice. During the early phases of infection, all three knockout mice had low Ag-specific IFN-gamma responses. However, this reduced IFN-gamma response was overcome during a state of persistent Ag stimulation (chronic infection), and was not associated with an adverse parasitologic outcome in any of the gene-targeted mouse strains. To the contrary, during the late phase of infection, an exaggerated Ag-specific IFN-gamma response was evident in CCR5-/- and MIP-1 alpha-/- mice, and this correlated with an enhanced control of parasite replication. Although granuloma formation was abnormal in each of the knockout mice, there was no correlation between the number or architecture of the granulomas and parasite burden. Collectively, these findings indicate an important role for CCR5, MIP-1 alpha, and CCR2 in granulomatous inflammation, and that CCR5 and MIP-1 alpha, possibly acting through CCR5, might play a deleterious role in the outcome of chronic L. donovani infection. Our data also suggest that there might be cross-talk between TCR and chemokine receptor signaling pathways.

Dendritic cell (DC)-based anti-infective strategies: DCs engineered to secrete IL-12 are a potent vaccine in a murine model of an intracellular infection.

Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-gamma, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1-3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-gamma response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies.

Activation and adherence of lyophilized human platelets on canine vessel strips in the Baumgartner perfusion chamber.

The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.

Mechanical characteristics of dilated polytetraflouroethylene used for transluminally placed endovascular grafts.

This study was conducted to assess the mechanical characteristics of dilated polytetraflouroethylene (PTFE) for use in transluminally placed endovascular grafts (TPEGs). Ten-centimeter lengths of 3- and 4-mm thinwalled PTFE were dilated to 8, 10, 12, and 15 mm diameters (3 mm) and 10-, 14-, 16-, and 20-mm diameters (4 mm), respectively (n = 6 for each size). The dilated PTFE segments were evaluated for leakage, further dilation, structural changes (with electron microscopy), and changes in wall thickness occurring after 24 hours of perfusion at pressures of 300-350 mmHg. Both 3- and 4-mm thinwalled PTFE could be dilated to five times their initial diameter before rupture occurred. Three-millimeter grafts dilated to 12- and 4-mm grafts dilated to 14 mm remained resistant to leakage at perfusion pressures up to 350 mmHg. When 3-mm grafts were dilated to 15 mm, the PTFE leaked saline at a rate of 20.3 +/- 9.3 cc per hour at 300 mmHg. pressure. Four-millimeter grafts dilated to 16- and 20-mm diameters leaked saline at 8.4 +/- 7.8 and 52.8 +/- 22 cc per minute, respectively, at the same pressure. No grafts were found to increase in diameter after 24 hours of pressure perfusion. Electron microscopy revealed that PTFE node size was significantly smaller in dilated grafts than in undilated grafts, but there was no significant change in internodal distance. This data suggests that thinwalled PTFE can be dilated to large diameters and retain sufficient strength to resist supraphysiologic pressures. Long-term studies are needed to determine the late structural integrity of dilated PTFE.

Characterization of a new hereditary thrombopathy in a closed colony of Wistar rats.

A new hereditary thrombopathy has been identified in a closed colony of Wistar rats. A simple and reproducible cuticle bleeding time test was developed as a rapid screening procedure for the bleeding diathesis. Affected animals exhibit markedly prolonged bleeding times and complete absence of platelet aggregation either with adenosine diphosphate (ADP) or with thrombin. Inheritance data suggest an autosomal dominant inheritance pattern with variable penetrance. Coagulation tests, platelet counts, plasma von Willebrand factor (vWF) activity, and clot retraction are within normal limits in thrombopathic animals. GPIb-dependent botrocetin-induced platelet agglutination was present in washed thrombopathic rat platelets. No discernible abnormality of intraplatelet organelles or granules was seen by transmission electron microscopy of thrombopathic platelets. A qualitative morphologic assessment of intraplatelet fibrinogen in thrombopathic rat platelets showed no discernible difference as compared with control rat platelets. Thrombopathic rat platelets exhibit decreased glycoprotein IIb/IIIa (GPIIb/IIIa) antigen by flow cytometric analysis and markedly decreased iodine 125-labeled fibrinogen binding to platelet GPIIb/IIIa after ADP activation. This rat colony demonstrates a unique thrombopathy, distinct from previously described animal thrombopathies, with some characteristics of variant Glanzmann's thrombasthenia. This animal model may provide further insight into the regulatory mechanisms and pathophysiology of platelet GPIIb/IIIa.

Pathology and laboratory medicine.

Automation streamlines a previously labor-intensive process and virtually eliminates sampling or labeling errors. The techniques of molecular biology and genetics continue to assist the physician in the diagnosis, prognosis, and treatment of patients with neoplastic diseases.

Nasopharyngeal nonossifying variant of ossifying fibromyxoid tumor: CT and MR findings.

The CT and MR findings of a nasopharyngeal nonossifying variant of an ossifying fibromyxoid tumor are presented. The findings are radiographically indistinguishable from more common malignant neoplasms encountered in this region. The tumor was isointense with muscle on T1-weighted images. On proton density- and T2-weighted images, the mass was mostly isointense with gray matter but contained some areas of lower intensity that might reflect the fibrous tissue component. The tumor eroded through the floor of the middle cranial fossa.

Function of von Willebrand factor after crossed bone marrow transplantation between normal and von Willebrand disease pigs: effect on arterial thrombosis in chimeras.

von Willebrand factor (vWF) is essential for the induction of occlusive thrombosis in stenosed and injured pig arteries and for normal hemostasis. To separate the relative contribution of plasma and platelet vWF to arterial thrombosis, we produced chimeric normal and von Willebrand disease pigs by crossed bone marrow transplantation; von Willebrand disease (vWD) pigs were engrafted with normal pig bone marrow and normal pigs were engrafted with vWD bone marrow. Thrombosis developed in the chimeric normal pigs that showed normal levels of plasma vWF and an absence of platelet vWF; but no thrombosis occurred in the chimeric vWD pigs that demonstrated normal platelet vWF and an absence of plasma vWF. The ear bleeding times of the chimeric pigs were partially corrected by endogenous plasma vWF but not by platelet vWF. Our animal model demonstrated that vWF in the plasma compartment is essential for the development of arterial thrombosis and that it also contributes to the maintenance of bleeding time and hemostasis.

Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption.

With the aim of establishing whether a genetically reduced capability of producing apolipoprotein E (apo E) can affect atherogenesis, we have compared the consequences of dietary stress on normal mice and on mice heterozygous or homozygous for a disrupted apo E gene. A dramatically accelerated development of lesions occurred in the vasculature of the homozygous mutants as a result of feeding an atherogenic diet for 12 wk, and extensive deposition of lipid-filled macrophages was found outside the cardiovascular system. In nine heterozygotes fed the atherogenic diet for 12 wk, the amount of apo E in their total plasma lipoproteins increased to a level comparable to normal, but all nine developed much larger foam cell lesions in their proximal aorta than those found in 3 of 9 normal mice fed the same diet. The other six normals had no lesions. Our study demonstrates that heterozygous mice with only one functional apo E gene are more susceptible to diet-induced atherosclerosis than are normal, two-copy mice. Genetically determined quantitative limitations of apo E could, therefore, have similar effects in humans when they are stressed by an atherogenic diet.

Salivary gland anlage tumor ("congenital pleomorphic adenoma"). A clinicopathologic, immunohistochemical and ultrastructural study of nine cases.

Salivary gland anlage tumor (SGAT) is a polypoid lesion of the nasopharynx that presents with respiratory distress at birth or within the first few days or weeks of life. Among our nine cases, there was a male predilection (7M:2F). All tumors were in the midline and attached to the posterior pharyngeal wall by a delicate pedicle. The largest tumor measured 3 cm. A biphasic histologic pattern of squamous nests and duct-like structures at the periphery blended into solid, predominantly mesenchymal-appearing nodules centrally. The surrounding submucosal mantle of epithelial structures was consistently immunoreactive for cytokeratin and epithelial membrane antigen, whereas the stromal-like cells of the central nodules showed variable immunopositivity for cytokeratin, vimentin, and muscle-specific actin. Both components were equally reactive for salivary gland amylase. Ultrastructurally, some of the stromal-like cells had features of myoepithelial cells. The histologic and architectural features of SGAT are similar in some respects to the developing salivary gland. It is proposed that the SGAT is a probable hamartoma of minor salivary gland derivation whose origin in the nasopharynx is potentially life-threatening in an infant.

Cadaver lung donors: effect of preharvest ventilation on graft function.

The pulmonary donor pool would increase substantially if lungs could be safely transplanted after cessation of circulation. To determine whether ventilation of cadaver lungs could improve graft function, canine donors were sacrificed and then ventilated with 100% oxygen (n = 6) or 100% nitrogen (n = 6); 6 served as nonventilated controls. Four hours after death, the lungs were flushed with modified Euro-Collins solution and harvested. Controls were ventilated with 100% oxygen only during flush and harvest. Recipients were rendered dependent on the transplanted lung by occlusion of the right pulmonary artery and bronchus 1 hour after transplantation. Ventilation was maintained at a constant inspired oxygen fraction of 0.4. Four controls died of pulmonary edema shortly after occlusion of the native lung. The mean arterial oxygen tensions in the oxygen-ventilated, nitrogen-ventilated, and control groups at the end of 8 hours were 81 mm Hg (n = 4), 88 mm Hg (n = 3), and 55 mm Hg (n = 2), respectively. Postmortem oxygen ventilation improved early recipient survival and gas exchange. Postmortem nitrogen ventilation improved early gas exchange and delayed recipient death compared with non-ventilated controls. The mechanics of ventilation appears to confer a functional advantage independent of a continued supply of oxygen. Transplantation of lungs harvested from cadavers after cessation of circulation might be feasible.

Prognostic factors in the recurrence of stage I and II squamous cell cancer of the oral cavity.

Stage I and II squamous cell cancers of the oral cavity have a high recurrence rate given their size and relative amenability to surgical resection. It has been suggested that one way to decrease this recurrence rate is to augment the surgical resection of these tumors with either elective neck dissection or radiation therapy. However, this would expose a significant number of patients to the unnecessary morbidity associated with either of these modalities. In an attempt to identify those patients most at risk for recurrence, we retrospectively determined the clinical and histologic factors that were associated with recurrence in 49 patients with stage I and II oral cavity cancer. Multiple regression analysis revealed that when various interactions between variables were controlled for, only the presence of a positive surgical margin or a tumor depth greater than 5 mm was significantly associated with recurrence. Each individually increased the likelihood of recurrence almost threefold.

Verruciform xanthoma in association with discoid lupus erythematosus.

Verruciform xanthoma (VX) is an uncommon lesion occurring primarily in the oral cavity. Cutaneous lesions are much less common and they preferentially arise on anogenital skin. They are not necessarily associated with a pre-existing inflammatory process. We report a VX in association with a long-standing lesion of discoid lupus erythematosus (DLE) on the scalp of a 34-year-old black woman. This association, which to our knowledge has not been previously reported, is consistent with the proposed pathogenetic mechanism of entrapment and subsequent degeneration of epithelial cells in the papillary dermis of VX. Histological distinction of VX from squamous cell carcinoma, with which this lesion may be clinically confused, is straightforward.