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Context Mutations in genes encoding for the succinate dehydrogenase (SDH) complex are linked to hereditary paraganglioma syndromes. Paraganglioma syndrome 3 is associated with mutations inSDHC and typically manifests as benign, nonfunctional head and neck paragangliomas. Design We describe a case of a 51-year-old woman who initially presented with diarrhea and hypertension and was found to have a retroperitoneal mass, which was resected with a pathology consistent with paraganglioma. Five years later, her symptoms recurred, and she was found to have new retroperitoneal lymphadenopathy and lytic lesions in the first lumbar vertebral body and the right iliac crest, which were visualized on CT scan and octreoscan but not on iodine-123-meta-iodobenzylguanidine (123I-MIBG) and bone scans. She had significantly elevated 24-hour urine norepinephrine and dopamine. The patient received external beam radiation and a series of different antineoplastic agents. Her disease progressed, and she eventually expired within 2 years. Genetic testing revealed a heterozygousSDHC c.43C>T, p.Arg15X mutation, which was also detected in her daughter and her grandson, both of whom have no biochemical or imaging evidence of paraganglioma syndrome yet. Conclusion We report a unique case of functional, metastatic abdominal paraganglioma associated withSDHC germline mutation. Our case exemplifies thatSDHC germline mutation has variable penetrance, which may manifest with an aggressive biology that could be missed by a123I-MIBG scan.

// Tai Qin 1, 2 , Lindsey Barron 1 , Lu Xia 1, 3 , Haojie Huang 5 , Maria M. Villarreal 4 , John Zwaagstra 9 , Cathy Collins 9 , Junhua Yang 1 , Christian Zwieb 4 , Ravindra Kodali 8 , Cynthia S. Hinck 8 , Sun Kyung Kim 4 , Robert L. Reddick 6 , Chang Shu 2 , Maureen D. O’Connor-McCourt 9 , Andrew P. Hinck 8 , Lu-Zhe Sun 1, 7 1 Department of Cell Systems and Anatomy, University of Texas Health Science Center, San Antonio, TX, USA 2 Department of Vascular Surgery, Second Xiangya Hospital and Xiangya School of Medicine, Central South University, Hunan, China 3 Department of Gynecology and Obstetrics, Xiangya Hospital and Xiangya School of Medicine, Central South University, Hunan, China 4 Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX, USA 5 Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA 6 Department of Pathology, University of Texas Health Science Center, San Antonio, TX, USA 7 Cancer Therapy and Research Center, University of Texas Health Science Center at San Antonio, Texas, USA 8 Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA USA 9 National Research Council Human Health Therapeutics Portfolio, Montreal, Quebec, Canada, Maureen O'Connor-McCourt is currently affiliated with Formation Biologics, Montreal, Quebec, Canada Correspondence to: Lu-Zhe Sun, email: SUNL@uthscsa.edu Andrew P. Hinck, email: ahinck@pitt.edu Keywords: TGF-β trap, RER, tumorigenesis, Pten, prostate cancer Received: August 15, 2016 Accepted: November 07, 2016 Published: November 14, 2016 ABSTRACT The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O6-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 μmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O6-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma. Cancer Prev Res; 9(3); 245–52. ©2015 AACR.

// Izhar Batth 1, 8 , Huiyoung Yun 2 , Suleman Hussain 2 , Peng Meng 1, 7 , Pawel Osmulski 3 , Tim Hui-Ming Huang 3, 5 , Roble Bedolla 1 , Amanda Profit 4 , Robert Reddick 4 , Addanki Kumar 1, 2, 3, 5, 6 1 Department of Urology, The University of Texas Health Science Center, San Antonio, TX, USA 2 Department of Pharmacology, The University of Texas Health Science Center, San Antonio, TX, USA 3 Department of Molecular Medicine, The University of Texas Health Science Center, San Antonio, TX, USA 4 Department of Pathology, The University of Texas Health Science Center, San Antonio, TX, USA 5 Cancer Therapy and Research Center, The University of Texas Health Science Center, San Antonio, TX, USA 6 The University of Texas Health Science Center at San Antonio and South Texas Veterans Health Care System, San Antonio, TX, USA 7 Current address: Life Sciences Division, Lawrence Berkley National Laboratory, Berkley, CA, USA 8 Current address: Department of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Addanki Kumar, e-mail: kumara3@uthscsa.edu Keywords: castrate resistant prostate cancer, apoptosis, FLIP, RON, MST1R Received: November 18, 2015 Accepted: January 29, 2016 Published: February 9, 2016 ABSTRACT Castrate-resistant prostate cancer (CRPC) is the fatal form of prostate cancer. Although reactivation of androgen receptor (AR) occurs following androgen deprivation, the precise mechanism involved is unclear. Here we show that the receptor tyrosine kinase, RON alters mechanical properties of cells to influence epithelial to mesenchymal transition and functions as a transcription factor to differentially regulate AR signaling. RON inhibits AR activation and subset of AR-regulated transcripts in androgen responsive LNCaP cells. However in C4-2B, a castrate-resistant sub-line of LNCaP and AR-negative androgen independent DU145 cells, RON activates subset of AR-regulated transcripts. Expression of AR in PC-3 cells leads to activation of RON under androgen deprivation but not under androgen proficient conditions implicating a role for RON in androgen independence. Consistently, RON expression is significantly elevated in castrate resistant prostate tumors. Taken together our results suggest that RON activation could aid in promoting androgen independence and that inhibition of RON in combination with AR antagonist(s) merits serious consideration as a therapeutic option during hormone deprivation therapy.

The purpose of this investigation was to compare the antitumorigenic effects of the natural product Nexrutine to voluntary wheel running (VWR) in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Forty-five, 10-week old TRAMP mice were randomized to either receive free access to the running wheel, Nexrutine pelleted into chow at 600 mg/kg or no treatment control. Mice were serially sacrificed at weeks 4, 8,12 and 20 weeks. Palpable tumors, body weight, food consumption and running wheel activity were monitored weekly. At necropsy, tumors and serum were harvested and stored for analysis. Serum was used to quantify circulating cytokines in 4 and 20 week time points. Nexrutine supplementation led to a 66% protection against high grade tumors. Exercise resulted in a 60% protection against high grade tumors. Both interventions reduced concentrations of IL-1α. Exercise also significantly lowered concentrations of eotaxin, IL-5, IL-12(p40) and VEGF. While there were no significant differences at baseline, exercise mice had significantly lower IL-5 and VEGF compared to control at the 20 week time point. Nexrutine also significantly reduced circulating IL-9 concentrations. No significant differences were observed when compared to the control group. Immunohistochemistry of tumor sections showed significantly lower expression of pAkt in Nexrutine fed mice with no visible differences for NFκB. In conclusion, both Nexrutine and exercise suppressed tumor growth. Though similar outcomes were seen in this comparative effectiveness study, the mechanisms by which exercise and Nexrutine exert this benefit may focus on different pathways.

Nuclear factor (NF)κB is a transcription factor that controls immune and inflammatory signaling pathways. In skeletal muscle, NFκB has been implicated in the regulation of metabolic processes and tissue mass, yet its affects on mitochondrial function in this tissue are unclear. To investigate the role of NFκB on mitochondrial function and its relationship with muscle mass across the life span, we study a mouse model with muscle-specific NFκB suppression (muscle-specific IκBα super-repressor [MISR] mice). In wild-type mice, there was a natural decline in muscle mass with aging that was accompanied by decreased mitochondrial function and mRNA expression of electron transport chain subunits. NFκB inactivation downregulated expression of PPARGC1A, and upregulated TFEB and PPARGC1B. NFκB inactivation also decreased gastrocnemius (but not soleus) muscle mass in early life (1–6 months old). Lower oxygen consumption rates occurred in gastrocnemius and soleus muscles from young MISR mice, whereas soleus (but not gastrocnemius) muscles from old MISR mice displayed increased oxygen consumption compared to age-matched controls. We conclude that the NFκB pathway plays an important role in muscle development and growth. The extent to which NFκB suppression alters mitochondrial function is age dependent and muscle specific. Finally, mitochondrial function and muscle mass are tightly associated in both genotypes and across the life span.

AIM/HYPOTHESIS: Endothelial dysfunction due to impaired endothelial nitric oxide synthase (eNOS) activity is a key factor implicated in the progression of diabetic nephropathy. Previous studies demonstrated that global deficiency of eNOS in diabetic mice exacerbated renal lesions. In this study we investigated whether targeted expression of eNOS and enhanced bioavailability of nitric oxide in renal microvascular endothelial cells rescues diabetic nephropathy. In transgenic mice, overexpression of eNOS has been shown to protect against tissue injury; however, the effect of targeted expression of eNOS in the kidney microvasculature has not been examined. METHODS: To determine if overexpression of eNOS in endothelial cells of diabetic mice ameliorates renal lesions, transgenic mice selectively expressing eNOS in endothelial cells (eNOSTg) were cross bred with Ins2Akita type-1 (AK) diabetic mice to generate eNOSTg/AK mice. Kidneys from wild type, eNOSTg, AK and eNOSTg/AK mice were assessed for morphology and reactive oxygen species production; each group was analyzed for blood glucose levels, kidney/body weight and urine albumin/creatinine levels. RESULTS: AK and eNOSTg/AK mice were hyperglycemic. eNOSTg mice unexpectedly showed evidence of glomerular injury with segmental mesangiolysis and occasional microaneurysms. As expected, glomeruli of AK mice showed diabetic changes with matrix expansion and early nodules. Notably, overexpression of eNOS in eNOSTg/AK mice led to increased glomerular/endothelial injury that was associated with increased superoxide levels and renal dysfunction. Results indicate for the first time that overexpressing eNOS in endothelial cells cannot ameliorate diabetic lesions, but paradoxically leads to progression of nephropathy likely due to the superoxide generated by the uncoupling of eNOS. CONCLUSION/INTERPRETATION: The eNOSTg/AK model will be useful for elucidating mechanisms that regulate eNOS function and may suggest therapeutic strategies to improve endothelial function and prevent progression of diabetic renal disease.

SIRT1 is a NAD+ dependent deacetylase known to regulate a plethora of biological processes through posttranslational regulation of proteins including those that function as tumor promoters and suppressors. In order to define the role of SIRT1 in prostate pathogenesis, we used 2 mouse models: (i) PTEN knockout (PTENKO) mouse model by pharmacological activation of SIRT1 with resveratrol (RES) a known activator of SIRT1, & (ii) orthotopic implantation model with genetic silencing of SIRT1 (SIRT1 shRNA). We also used genetic and pharmacologic inhibition of SIRT1 in cell culture models to understand the mechanism. We tested whether SIRT1 modulation is beneficial when targeted early (before the establishment of prostatic lesions) or late (after the establishment of prostatic lesions) in the PTENKO model. RES intervention was initiated in 4-5-week (early intervention) and 10-15-week-old (late intervention) PTENKO mice. Analyses of samples collected longitudinally during progression revealed that early intervention with RES reduced incidence of high-grade prostate intraepithelial neoplastic lesions (HGPIN) when given for 14 weeks with no significant difference at 7 or at 11 weeks. On the other hand, late intervention after the establishment of HGPIN lesions with RES had no beneficial effect. Importantly, longer treatment duration (28 weeks) resulted in significantly increased incidence of invasive prostate carcinoma. Furthermore, RES had no significant effect on the development of orthotopic prostate tumors following implantation of LNCaP cells in nude mice. In contrast, orthotopic implantation of SIRT1 stably silenced LNCaP cells showed significant impairment in tumor development. Immunohistochemical evaluation showed nuclear localization of SIRT1 in human prostate tumors and was associated with increased risk of biochemical recurrence. Mechanistic investigations revealed (i) suppression of AR signaling in hormone-sensitive LNCaP but not in castration-resistant 22Rv1 cells; (ii) RNAseq coupled with gene ontology enrichment analysis using SIRT1 silenced cells under conditions of androgen stimulation and inhibition identified genes involved in cell cycle checkpoint and senescence as top pathways affected by SIRT1 loss of function. In silicoanalysis shows that the identified SIRT1-regulated targets are associated with disease aggressiveness and poor disease-free survival. Taken together these data demonstrate that (i) SIRT1 plays a contextual role during prostate pathogenesis by functioning as tumor suppressor during early stage and as a tumor promoter during late stage; (ii) SIRT1 inhibition suppresses tumor development and (iii) RES is a better preventive than therapeutic agent. Therefore, our findings offer promising avenues to develop SIRT1-regulated pathways as novel therapeutic targets to inhibit prostate cancer recurrence. Supported by CPRIT Training Grant RP 170345 (SH) and CPRIT RP 150166 (APK) Citation Format: Shih-Bo Huang, Dinesh Thapa, Roble G. Bedolla, Amanda R. Muñoz, Xiaoyu Yang, Paul Rivas, Robert L. Reddick, Hiroshi Miyamoto, Rita Ghosh, Addanki Pratap Kumar. SIRT1 functions as a double-edged sword in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5082.

Myriad infectious organisms can infect the endocardium, myocardium, and pericardium, including bacteria, fungi, parasites, and viruses. Significant cardiac infections are rare in the general population but are associated with high morbidity and mortality as well as increased risk in certain populations, such as the elderly, those undergoing cardiac instrumentation, and intravenous drug abusers. Diagnostic imaging of cardiac infections plays an important role despite its variable sensitivity and specificity, which are due in part to the nonspecific manifestations of the central inflammatory process of infection and the time of onset with respect to the time of imaging. The primary imaging modality remains echocardiography. However, cardiac computed tomography and magnetic resonance (MR) imaging have emerged as the modalities of choice wherever available, especially for diagnosis of complex infectious complications including abscesses, infected prosthetic material, central lines and instruments, and the cryptic manifestations of viral and parasitic diseases. MR imaging can provide functional, morphologic, and prognostic value in a single examination by allowing characterization of inflammatory changes from the acute to chronic stages, including edema and the patterns and extent of delayed gadolinium enhancement. We review the heterogeneous and diverse group of cardiac infections based on their site of primary cardiac involvement with emphasis on their cross-sectional imaging manifestations. Online supplemental material is available for this article. (©)RSNA, 2016.

Over the last 30 years, little improvement has been made to the 5 year survival rate of pancreatic cancer (PanCA) patients. While survival has increase by 2% over the last several years, the current rate is still less than 8%. This depressing fact demonstrates the importance of developing or improving therapies to more effectively manage this disease. Along these lines, published studies from our laboratory demonstrated the anti-tumorigenic potential of the cork tree bark extract, Nexrutine® (Nx). Nx suppressed growth of pancreatic cancer cells through downregulation of STAT3/NF-κB activation. Subsequent biochemical and molecular investigations revealed palmatine (PMT) (i) as an active constituent of Nx able to suppress the growth of pancreatic cancer cells; (ii) synergizes with gemcitabine (GEM); and (iii) downregulates GLI1, COL1A1 and Survivin. Despite such promising in vitro observations however, the in vivo relevance of PMT is undefined. Furthermore, it is unclear if PMT can recapitulate the biological activities of Nx in vivo. In this investigation, we tested the hypothesis that PMT recapitulates the biological activities of Nx and enhances GEM activity. This hypothesis was tested by comparing the efficacy of Nx and PMT using (i) athymic mice implanted with Capan-2 cells; (ii) a syngenic mouse model using C57BL/6 mice implanted with KPC-GFP-Luc cells; and (iii) a short term ex vivo model utilizing cells isolated from primary pancreatic tumors following surgical resection. Additionally, in vitro experiments were also done to assess the underlying molecular mechanism. Analysis of these data show that both Nx and PMT are well tolerated in vivo and a significant reduction in the levels of serum inflammatory cytokines including IL-6, granulocyte-colony stimulating factor (G-CSF), and CXCL1. Interestingly, animals receiving PMT, but not Nx, showed a trend towards decreased pancreatic tumor weight that was associated with histopathological changes. Investigation into the potential mechanism revealed that Nx and PMT mediated inhibition of STAT3, EP4, Src, TrkA, and RPS6 activities may contribute to the observed growth inhibitory and anti-inflammatory effects. Incredibly, our ex vivo analysis of patient derived PanCA cells demonstrated that both Nx and PMT could inhibit the growth of these cells. Collectively, our data demonstrates PMT recapitulates biological activities of Nx and that there is potential for developing PMT as an agent for clinical management of PanCA. Supported by NCCIH (R01 AT007448; APK) and VA-MERIT Award (I01 BX 000766; APK). Citation Format: Amanda R. Muñoz, Roble Bedolla, Shih-Bo Huang, Xiaou Yang, Paul Rivas, Robert Reddick, Martha Hanes, Glenn Halff, Rita Ghosh, Addanki P. Kumar. Palmatine as a potential pancreatic cancer therapeutic agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5823.

Background: Elderly individuals react less efficiently to vaccines than do adults, mainly because of T-cell unresponsiveness. In this study, we analysed whether tumour-associated antigen (TAA)-specific CD8 T-cell responses could be induced by vaccination in old mice with metastatic breast cancer. Methods: The effect of pcDNA-3.1- and Listeria-based vaccines, expressing TAA Mage-b, on Mage-b-specific immune responses was tested in spleens and draining lymph nodes (LNs) of mild (4TO7cg) and aggressive (4T1) syngeneic metastatic mouse breast tumour models at young (3 months) and old (20 months) age. Results: Interferon γ and interleukin-2 levels increased significantly in draining LNs and spleens of Mage-b-vaccinated mice compared with those in control groups at young but not old age in both mouse tumour models. A significant increase was observed in the number of IFNγ-producing Mage-b-specific CD8 T cells after Mage-b vaccination in the 4T1 model at young but not old age. This correlated with a reduced protective effect of Mage-b vaccination against metastatic breast cancer at old compared with young age. Conclusions: The absence of CD8 T-cell responses after Mage-b vaccination and the accompanying reduced protection against metastatic breast cancer in old compared with young mice point towards the need for tailoring cancer vaccination to older age.

BACKGROUND Transgenic adenocarcinoma of mouse prostate (TRAMP) mice, derived by prostate specific expression of SV40 large T antigen using the rat probasin promoter, all develop prostate tumors akin to human prostate cancers. More recently, epithelial–stromal (ES) tumors resembling phyllodes tumors have been described in the seminal vesicles of TRAMP mice. We report malignancy arising in these ES tumors of the seminal vesicles in TRAMP mice. METHODS H&E stained sections from 28-week-old TRAMP mice autopsies were examined. Immunostains (cytokeratin, vimentin, desmin, and MIB-1) and electron microscopy were performed on selected blocks of the genitourinary system and metastatic tumor nodules. RESULTS The seminal vesicles frequently develop tumors containing broad papillae, with bland epithelium and a cellular spindled stroma just beneath the epithelium. The stromal cells have high nuclear to cytoplasmic ratio, frequent apoptotic cells and mitoses. In some cases, the stromal cells become large mass lesions that overgrow the prostate. The epithelium can also proliferate and become malignant. The tumors have high proliferation indices by MIB-1. Some metastatic tumors have characteristics similar to the seminal vesicle ES tumor. CONCLUSIONS Metastatic tumors in TRAMP mice show three patterns: (1) A definite adenocarcinoma pattern metastatic from the prostate; (2) poorly differentiated tumor without epithelial differentiation; (3) carcinosarcomatous pattern. The carcinosarcomatous pattern and some of the poorly differentiated tumors likely arise from seminal vesicle ES tumors. Prostate 69: 755–760, 2009. © 2009 Wiley-Liss, Inc.

Functional consequences of impaired endothelial nitric oxide synthase (eNOS) activity causing organ-specific abnormalities on a diabetic setting are not completely understood. In this study, we extensively characterized a diabetic mouse model (lepr(db/db)) in which eNOS expression is genetically disrupted (eNOS-/-). The eNOS-/-/ lepr(db/db) double-knockout (DKO) mice developed obesity, hyperglycemia, hyperinsulinemia and hypertension. Analysis of tissues from DKO mice showed large islets in the pancreas and fat droplets in hepatocytes. Interestingly, the aorta was normal and atherogenic lesions were not observed. Abnormalities in the aorta including poor re-endothelialization and increased medial wall thickness were evident only in response to deliberate injury. In contrast, significant glomerular capillary damage in the kidney was identified, with DKO mice demonstrating a robust diabetic nephropathy similar to human disease. The vascular and renal impairments in DKO mice were pronounced despite lower fasting plasma glucose levels compared to lepr(db/db) mice, indicating that eNOS is a critical determinant of hyperglycemia-induced organ-specific complications and their severity in diabetes. Results provide the first evidence that absence of eNOS in diabetes has a greater deleterious effect on the renal microvasculature than on the larger aortic vessel. The DKO model may suggest novel therapeutic strategies to prevent both vascular and renal complications of diabetes.

The host factors that influence autoimmune arthritides such as rheumatoid arthritis have not been fully elucidated. We previously found that genetic inactivation of CC chemokine receptor 2 (CCR2) in the arthritis-prone DBA/1j mouse strain significantly increases the susceptibility of this strain to autoimmune arthritis induced by immunization with collagen type II (CII) and complete Freund's adjuvant (CFA). Here, we show that following intradermal infection with Mycobacterium avium, a similar arthritis phenotype was detected in Ccr2-null mice in the DBA/1j, but not in the BALB/c background. The failure to develop arthritis in Ccr2-null BALB/c mice occurred in the face of high bacterial burdens and low interferon gamma (IFNgamma) production. By contrast, Ccr2-null DBA/1j mice had low bacterial burdens, produced normal amounts of IFNgamma, and had high titers of autoantibodies against CII. Thus, the Ccr2-null state in an arthritic-prone genetic background leads to increased arthritis susceptibility following infectious (M. avium) and noninfectious (CII/CFA) challenges. Because CCR2 serves as a negative regulator of murine arthritis, caution might need to be exercised while testing CCR2 blockers in human arthritis or other diseases. These findings also indicate that Ccr2-null DBA/1j mice might serve as a valuable model system to uncover the immunological determinants of arthritis and to test novel antiarthritic agents.

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate Various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tomor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4+ and CD8+ T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat Pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors Were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8+ T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8+ T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20–22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

With a 5 year survival of less than 8%, the poor prognosis of pancreatic cancer (PanCA) underlines the importance of improving therapies for effective management of this disease. In previous studies, we identified the tumor cell growth inhibitory activities for Nexrutine® (Nx, a bark extract from Phellodendron amurense) and palmatine (PMT) which is a constituent of Nx. These studies revealed an important role for downregulation of KRAS downstream effectors including GLI, STAT3 and NF-κB in mediating growth inhibitory effects. Remarkably, both Nx and PMT exerted synergistic growth inhibitory effects in combination with the conventional chemotherapeutic agent, Gemcitabine (GEM). Despite such promising in vitro observations, the in vivo efficacy of Nx or PMT had not been established. In this investigation, we evaluated the ability of Nx and PMT to (i) prevent the development of pancreatic tumors; and (ii) inhibit the growth of patient derived pancreatic cancer tissue in short term ex vivo cultures. Studies were also conducted to investigate the underlying molecular mechanism using cell culture models. Our results show that both Nx and PMT are well tolerated in vivo as evidenced by a lack of significant changes in the body weights of athymic mice implanted with Capan-2 cells. Interestingly, both Nx and PMT showed a significant decrease in the circulating levels of inflammatory molecules including IL-6 and CXCL1. In this first proof-of-concept study, PMT treated animals demonstrated a trend towards decreased pancreatic tumor weight with associated histopathogical changes. Remarkably, both Nx and PMT inhibited the growth of patient derived pancreatic cancer cells. Mechanistic investigations identified downregulation of STAT3, TrkA, Src, and RPS6 activities as potential contributors to Nx or PMT-induced synergistic growth inhibitory effects with GEM. Further investigation of STAT3 signaling revealed the involvement of the prostaglandin receptor, EP4, in a potential feedback loop with STAT3 and in asserting STAT3-mediated inhibition of autophagy. Taken together these data support potential utility for Nx and/or PMT in the management of PanCA and suggest that these agents use multiple mechanisms to affect growth of pancreatic tumors. Supported by NCCIH (R01 AT007448; APK) and VA-MERIT Award (I01 BX 000766; APK). Citation Format: Amanda R. Munoz, Roble G. Bedolla, Paul Rivas, Divya Chakravarthy, Robert L. Reddick, Martha A. Hanes, Glenn A. Halff, Rita Ghosh, Addanki P. Kumar. Nexrutine and palmatine mediated effects on the STAT3/EP4/IL-6 axis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2017-5268

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

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.

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.

We have studied the roles of von Willebrand factor (vWF) and factor VIII in arterial thrombosis in four canine phenotypes: normal (n = 6), hemophilia A (n = 11), von Willebrand disease (vWD) (n = 9), and hemophilia A/vWD (n = 1). vWF activity was determined by botrocetin- induced agglutination of fixed human platelets and vWF antigen (vWF:Ag) by Laurell electroimmunoassay and crossed immunoelectrophoresis. Plasma from normal dogs and those with hemophilia A had vWF activity, vWF:Ag, and a full range of vWF:Ag multimers on gel electrophoresis equivalent to normal canine plasma pool. Platelet cytosol contents were isolated by freezing and thawing, triton X-100 solubilization, or sonication of washed platelets with and without protease inhibitors and inhibitors of platelet activation. Washed platelets were also stimulated with calcium ionophore and MgCl2. There was no measurable vWF activity or vWF:Ag in platelet lysates or releasates in any dog regardless of phenotype. All dogs were studied using a standard arterial stenosis and injury procedure to induce arterial thrombosis. Thromboses were detected by cyclic reductions in Doppler blood flow velocity. Vessels were examined by light and scanning electron microscopy. Thrombosis developed in the arteries of normal (9 of 10) and hemophilia A dogs (16 of 16) but in none of the vWD dogs (0 of 10). Infusion of canine vWF cryoprecipitate into vWD dogs markedly shortened bleeding time but did not support thrombosis as seen in dogs with vWF in the plasma and subendothelium. Thrombosis, then, fails to occur when vWF is absent from the plasma and subendothelial compartments or present only in the plasma compartment. These data are consistent with the hypothesis that vWF in the plasma and subendothelium supports thrombosis. Neither plasma FVIII nor platelet vWF is essential for thrombosis in this model.

A shortage of suitable donors is a serious obstacle to the widespread application of isolated lung transplantation for end-stage lung disease. We hypothesized that lung tissue likely remains viable for a sufficient period of time to allow for safe postmortem retrieval of lungs for transplantation. Studies were conducted in a nonsurvival model of canine lung allotransplantation. Donor animals were sacrificed, and subsequent lung harvest was delayed for 1 hour, 2 hours, or 4 hours. Pulmonary retrieval was then performed in a standard fashion, flushing the lung block with modified Euro-Collins solution. Lungs were then stored for 4 hours before single allotransplantation. Recipient animals were maintained anesthetized, and followed up for 8 hours. By occlusion of the pulmonary artery and bronchus to the native lung, recipient animals were forced to survive solely on the transplanted lung, with a constant inspired oxygen fraction of 0.40. All 5 recipient animals of 1-hour cadaver lungs survived the 8-hour observation period with excellent hemodynamics and gas exchange. Two of 5 recipients of 2-hour cadaver lungs survived the observation period, whereas a third animal survived for 5 hours with excellent gas exchange. One of 4 animals transplanted with a 4-hour cadaver lung survived the observation period. Retrieval of lungs from cadavers whose hearts are not beating may prove to be a safe and effective method to increase the pulmonary donor pool.

Publisher Summary Technologic advancements in the genetic modifications in mice that have occurred during the last decade have enabled the generation of multiple models that each exhibit conditions like human cardiovascular disease. Consequently, the mouse is now clearly established as the premier model for complex cardiovascular research. This chapter illustrates, with emphasis on atherosclerosis, how use of the mouse has contributed to the current knowledge of vascular disease pathogenesis. The pathology of atherosclerotic lesions developing in mice lacking apolipoprotein E or low-density lipoprotein receptor is very similar to that seen in human patients with one major limitation that the spontaneous hemorrhage and rupture associated with human plaques have not been observed reliably in mice. Genetic as well as pharmacologic approaches using the mouse models of atherosclerosis have consequently concentrated on the early stages of atherogenesis and have produced a wealth of information regarding the factors that modify the development of atherosclerosis. Surgically induced cardiac complications in the atherogenic mouse models have also been utilized to define how hyperlipidemia influences the pathogenesis of other cardiovascular diseases such as myocardial infarction and restenosis. Continued improvements can be expected in both the mouse model and the sensitivity and miniaturization of various diagnostic procedures used in their evaluation. Thus, the mouse continues to promise additional exciting and productive times in cardiovascular research.

Radiation Therapy (RT) is a definitive treatment for early-localized prostate cancer (PCA), but is associated with side effects which impair quality of life in addition to development of radioresistance. PI3K/Akt/mTOR signaling is one of the contributors to therapeutic resistance, including radioresistance. Ribosomal protein S6 (rpS6), a downstream effector of PI3K/Akt/mTOR signaling, mediates radioresistance by increasing protein synthesis, cell survival and epithelial mesenchymal transition. Also, increased activation of rpS6 is correlated with poor survival in PCA. Therefore, downregulation of rpS6 could decrease RT induced toxic side effects by sensitizing tumor cells. Based on published evidence demonstrating tumor growth inhibitory activities, we tested if Nexrutine® (Nx), an inexpensive OTC herbal supplement from Phellodendron amurense bark extract, could potentiate RT by inhibiting rpS6 activation. Using clonogenic assays, low dose RT in combination with Nx was found to have similar inhibition of surviving fraction compared to high dose RT in androgen independent PC-3 cells. Isobologram analysis of these data depicted strong synergism. In addition, increased activation of Akt/NFKB signaling molecules was observed in PC-3 cells exposed to radiation. The observed radiation-induced increase in these signaling molecules was either abolished (p-rpS6, p-NFKB and p-p70S6K) or decreased (p-Akt) in cells pretreated with Nx (8 hours) prior to RT. Additionally, Nx pretreatment prolonged the G2/M arrest caused by RT in PC-3 cells. Strikingly, knockdown of rpS6 in PC-3 cells reversed the observed effects of Nx, indicating the importance of rpS6 in mediating these changes. Furthermore, administration of Nx in combination with RT inhibited prostate tumor progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with no prominent toxicity. Notably, immunohistochemical analysis revealed decreased levels of p-mTOR, p70S6K, NFKB, Ki67 and Cyclin D1 in the prostate in combination group of animals compared to monotherapy. Taken together, our data suggest that Nx sensitizes PCA cells by down regulating rpS6 and delays progression to lethal disease. Remarkably, in a recent phase 0/1 study patients (81%) receiving Nx showed decreased PSA levels with no signs of grade 3 toxicity. Thus, Nx shows immense potential for use as an adjuvant in combination with conventional therapy for effective clinical management of PCA. Supported by NCCAM (R01 AT-007448) and VA-MERIT Award (I01 BX 000766; APK) Citation Format: Suleman S. Hussain, Paul Rivas, Roble G. Bedolla, Nikos Papanikolaou, Robert L. Reddick, Brad H. Pollock, Daniel C. Chan, Rita Ghosh, Addanki P. Kumar. Downregulation of ribosomal protein S6 overcomes radioresistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1797. doi:10.1158/1538-7445.AM2015-1797

Anti-tumor vaccines are a relatively non-toxic alternative to conventional chemotherapeutic strategies to control breast cancer. Immunization with tumor-associated antigens (TAAs) triggers anti-tumor cytotoxic T lymphocytes (CTL), which can limit tumor progression. Here we report on the development and effectiveness of a TAA-based DNA vaccine encoding Mage-b1/2, the mouse homologue of the human MAGE-B1/2. As model system, we used immune competent Balb/c mice with syngeneic non-metastatic (64pT) or metastatic (4TO7cg) breast tumors. First, the presence of Mage-btranscripts in the 64pT and 4TO7cg breast tumors and metastases was demonstrated by RT-PCR, Southern blotting, and DNA sequencing. A DNA-based vaccine was developed from transcripts of one of the 64pT tumors, encoding the complete Mage-b1/2 protein, and subsequently tested for its preventive efficacy in both breast tumor models. Mice were immunized two times intramuscularly with the vaccine (pcDNA3.1-Mage-b1/2-V5), the control vector (pcDNA3.1-V5), or saline. Two weeks after the last immunization, the syngeneic 4TO7cg or 64pT tumor cell lines were injected in a mammary fat pad. Mice were monitored during the next 4 weeks for tumor formation, latency and size, and subsequently sacrificed for analysis. While the Mage-b1/2 vaccine had only a minor effect on the latency and growth of primary tumors, a significant and reproducible reduction in the number of 4TO7cg metastases was observed (vaccine versus control vector, p=0.0329; vaccine versus saline, p=0.0128). The observed protective efficacy of the Mage-b DNA vaccine correlated with high levels of vaccine-induced IFNgamma in spleen and lymph nodes upon re-stimulation in vitro. These results demonstrate the potential of TAA-based DNA vaccines in controlling metastatic disease in breast cancer patients.

Despite considerable evidence suggesting that hypertension contributes to the development and progression of atherosclerosis, the causative links remain unclear. We have tested the effects of chronic hypertension induced by suprarenal aortic constriction on the development of atherosclerosis in apolipoprotein E–deficient ( Apoe −/− ) mice. Compared with a sham operation, narrowing the aortic luminal diameter by 33% increased blood pressure proximal to the constriction by ≈15 mm Hg, but the pressures distal to the constriction were unchanged. Kidney renin mRNA and plasma renin activity were also unaffected. Compared with plaque size after the sham operation, atherosclerotic plaque size in the aortic root 8 weeks after coarctation was increased to 245% and 152% in males and females, respectively. Aortic segments at the constriction were free of atherosclerotic deposits, but segments proximal to the constriction were dilated and had atherosclerotic lesions. Thrombi were present immediately below the constriction in Apoe −/− and wild-type vessels. Surprisingly, compared with wild-type mice, the Apoe −/− mice were more susceptible to the cardiac hypertrophy and dysfunction induced by pressure overload. Thus, aortic coarctation exacerbates atherosclerosis in vessels proximal to the constriction without a concomitant increase in the renin-angiotensin system. Our study also suggests that apolipoprotein E plays an important role in modulating cardiac hypertrophy.

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.

Dietary administration of probucol (0.5%, wt/wt) efficiently reduced total plasma cholesterol levels in apolipoprotein E-deficient mice (apoE-/-) by 40%, with decreases in high density lipoprotein (HDL) and apoAI by 70 and 50%, respectively. Paradoxically, however, aortic atherosclerotic plaques in the probucol-treated apoE-/- mice formed more rapidly than in the untreated apoE-/- mice, and the lesions were two to four times larger and more mature regardless of sex, age, and genetic background (P < 10(-)6). Histologically, lesions in probucol-treated mice contained increased fibrous materials and cells other than foam cells, and were commonly associated with focal inflammation and aneurysmal dilatation. Probucol treatment also accelerated lesion development in apoE+/- mice fed an atherogenic diet, indicating that the adverse effect is not dependent on the complete absence of apoE. Furthermore, mice lacking apoE and apoAI have plasma lipoprotein profiles very similar to the probucol-treated apoE-/- mice, but do not have accelerated plaque development. Thus, the enhanced atherosclerosis in the probucol-treated animals is unlikely to be caused by the reduction of HDL and apoAI levels. Our data indicate that a reduction in plasma cholesterol caused by probucol does not necessarily lead to an antiatherogenic effect.

A 3rd generation perfluorocarbon (PFC) is under development for the treatment of traumatic brain injury (TBI). PFCs have high gas solubility and can deliver oxygen to support ischemic brain tissue. This drug increased brain tissue oxygen consumption, decreased infarct size, and improved functional deficits in animal models of brain injury, but it also caused transient thrombocytopenia of unknown etiology. Acute inflammation also causes platelet deficits and is common in the setting of severe TBI. The interaction between the two could exacerbate thrombocytopenia. Here we studied PFC effects on platelet function, platelet morphology and global hemostasis in a lipopolysaccharide (LPS) baboon model of inflammation. Perfluorocarbon (Oxycyte”, Oxygen Biotherapeutics Inc, Morrisville, NC) infusion was performed with and without administration of LPS. The groups studied included: 1. Saline control (20 ml saline + 12 ml/kg saline: SALINE) 2. Saline and LPS control (0.3 mg/kg LPS in 20 ml saline + 12 ml/kg saline: SAL-LPS) 3. Medium dose PFC with saline vehicle (20 ml SALINE + 3 ml/kg PFC: SAL-PFC3) 4. High dose PFC with saline vehicle (20 ml SALINE + 12 ml/kg PFC: SAL-PFC12) 5. Medium dose PFC with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 3 ml/kg PFC: LPS -PFC3) 6. High dose PFC with with LPS (LPS 0.3 mg/kg in 20 ml SALINE + 12 ml/kg PFC: LPS –PFC12) Platelet count, transmission electron microscopy, and flow cytometry were performed at baseline (BL) and at 2, 24, 48, 72, and 96 hours after perfluorocarbon infusion. Microparticles were quantified and characterized by flow cytometry and morphology examined via transmission electron microscopy. Impedance aggregometry and thromboelastography (TEG) were quantified at baseline (BL) and at T2, T72, and T96 (hours). Statistical analysis was by ANOVA, followed by pairwise comparisons and a Bonferroni adjustment. PFCs caused delayed thrombocytopenia which began 2 days post-infusion and persisted beyond the 5-day period of observation (nadirs≤50% of baseline at day 5, p PFC infusion caused clinically significant thrombocytopenia, particularly after LPS administration, and exacerbated LPS-induced platelet activation. The interaction between the two resulted in decreased hemostatic capacity, diffuse bleeding, and shock. Disclosures: Shade: Oxygen Biotherapeutics, Inc.: Research Funding. Anderson:Oxygen Biotherapeutics, Inc.: Employment.

Recently we have shown that resveratrol (RES) intervention prevents development of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions in prostate-specific PTEN knockout mouse model targeting SIRT1/mTORC1 axis. It is known that insufficient nutrient supply combined with high proliferation keeps solid tumors including prostate under hypoxic and metabolic stress. Tumor cells adapt to survive under such conditions through activation of AMP-activated kinase (AMPK). AMPK has been reported to be activated in prostate tumors. Therefore targeting AMPK and associated signaling pathways will be a promising approach for prostate cancer management. Accordingly we investigated the role of AMPK in RES-induced growth inhibitory effects using multiple human prostate cancer cell lines and preclinical animal model. These data show that RES treatment (50 μM, 24 h) results in activation of SIRT1, significant inhibition of AMPK phosphorylation and cell survival in human prostate cancer cells (RWPE-1, LNCaP, C42B and DU145). The observed molecular changes were associated with induction of apoptosis and autophagy following treatment with RES. Further dietary administration of RES (0.1 and 2%) to 4-5 week old prostate-specific PTEN knockout mice for 11 and 14 weeks showed prevention of HGPIN development. Interestingly intervention for 7 weeks showed prevention of HGPIN development at lower dose but not at high dose. On the other hand, 28-week intervention had no significant effect on the development of HGPIN lesions. Immunohistochemical evaluation showed modulation of AMPK, pS6K and SIRT1 in the prostate. Overall these data provide novel insights into RES-induced prevention of HGPIN development via AMPK/SIRT1/mTORC1 axis. Supported in part by NIH (CA 137518 and 135451 APK). Citation Format: Guiming Li, Paul Rivas, Roble Bedolla, Robert L. Reddick, Rita Ghosh, Addanki P. Kumar. AMPK-SIRT1 axis: a potential therapeutic target for prostate cancer management. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2013-3673

Abstract Thrombotic thrombocytopenia with severe depletion of plasma von Willebrand factor (vWF) was induced in normal large animals (5 dogs and 2 pigs) by botrocetin, a Bothrops factor requiring vWF for platelet agglutination. Botrocetin (90 to 100 U/kg, 2.14 to 2.38 mg/kg, in a single IV injection) reduced plasma vWF activity to

Resveratrol (3,4′-trihydroxystilbene), a natural product is present in significant concentrations in red wine, peanuts, walnuts etc. Emerging evidence indicates that resveratrol exerts antitumorigenic activity in various tumor models including prostate. Although multiple biological effects on proliferation, apoptosis and activation of SIRT1 have been demonstrated to be associated with resveratrol-induced activities, the precise mechanism associated with resveratrol-induced SIRT1 activation and cancer cell growth inhibition remains unclear. In addition, whether resveratrol intervention suppresses the development and progression of PIN (Prostatic Intraepithelial Neoplasia) is unknown. In this study, we examined the role of SIRT1 and the underlying mechanism involved in resveratrol-mediated biological effects. Our results show that although resveratrol significantly inhibited proliferation of multiple prostate cell lines including RWPE-1, C42B, PC3 and DU145 at higher concentrations (> 50 µM); however, at lower concentrations (< 25 µM), resveratrol prompted cell proliferation. Interestingly androgen responsive LNCaP cells exhibited resistance to resveratrol-mediated inhibition of cell proliferation. Under these experimental conditions, we also observed significant increase in autophagy without remarkable increase in apoptosis. Given the role of Akt/mTOR signaling pathway as a negative regulator of autophagy, we investigated whether resveratrol induce autophagy via modulation of Akt/mTOR signaling. Our data indicate that resveratrol-induced autophagy was associated with decreased levels of mTORC1 activity and increased SIRT1 expression. Knockdown of SIRT1 enhanced prostate cell proliferation and attenuated resveratrol-induced autophagy. Using prostate specific PTEN−/− mice, we found that dietary administration of resveratrol reduced the incidence of high-grade PIN lesions significantly. These findings implicated an important role for mTORC1/SIRT1 signaling axis in mediating resveratrol-induced upregulation of autophagy and inhibition of apoptosis. [Supported by NIH CA137578 (APK)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2585. doi:1538-7445.AM2012-2585

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 nonventilated 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.

To explore and develop protocols for drying blood platelet preparations for extended storage without undue compromise of hemostatic function or cell integrity upon rehydration. A successful project outcome would greatly facilitate the use of platelets in the medical support of combat casualties with severe blood loss, major surgical repair, or extensive burn injury. The three collaborating performance sites are bringing together expertise in cell stabilization techniques and platelet function testing both in vitro and in vivo. The ultimate goal is to identify a practical procedure to produce a stabilized platelet preparation that can be stored virtually indefinitely while maintaining transfusion efficacy. Such a product would also be useful in civilian blood banking. The third year of this grant saw advancement of the animal model studies testing the hemostatic capabilities and circulatory survival of dried platelets stabilized with paraformaldehyde, refinement of an alternative platelet stabilization method using permanganate and trehalose, and an initial study of dried RBC in an in vivo circulatory model. In normal canine experimental subjects, rehydrated canine platelets were found in circulation for at least 24 hours after infusion; similar results were obtained in bleeder pigs (a congenital strain with von Willebrand's disease). Rehydrated platelets were shown to adhere to subendothelium in situ when the animal subjects were exposed to a vessel stenosis and injury protocol or subjected to a bleeding time test.

The biological behavior of pleomorphic adenomas (mixed tumors) of salivary gland origin is complex. Tumors with benign histologic features may exhibit recurrence and locally aggressive behavior especially after incomplete excision. A small percentage of pleomorphic adenomas have obvious malignant components in epithelial or in both epithelial and mesenchymal components and can metastasize. There are also rare case reports which appear to document typical pleomorphic adenomas of salivary gland with histologically identical visceral and lymph node metastases. Recently myoepithelial cell proliferation has been identified as a possible predictor of aggressive clinical behavior in otherwise histologically benign pleomorphic adenomas. We report such a parotid gland lesion with local recurrence and retroperitoneal spread. DNA-flow cytometry of cells from the paraffin-embedded primary and metastasis showed similar aneuploid populations. Aneuploidy appeared to reflect the malignant potential of this particular pleomorphic adenoma and suggests that DNA-flow cytometry of salivary gland tumors may yield important prognostic information.

The thrombogenic potential of atherosclerotic diet-induced coronary atherosclerotic plaques was investigated in normal swine and in bleeder swine with homozygous von Willebrand disease. Fourteen paired normal and bleeder swine were placed on a 1% cholesterol diet for 1 to 16 weeks. Serum cholesterol was elevated in all animals at sacrifice. Foam cell deposits developed in all major epicardial coronary arteries, and lesions progressed over time from small subendothelial foam cell deposits to fibrous cap lesions that contained foam cells, elastic fibers, collagen, degenerative material, and smooth muscle cells. Balloon catheter injury resulted in platelet deposition, largely in a monolayer or as small platelet clumps devoid of fibrin. Lipid debris was present in injured areas of the subendothelium but did not induce thrombus formation. When the injury involved the media, platelet-fibrin thrombi were formed. Lipid was not present in these thrombi. Morphometric analysis of platelet deposition on ballooned atherosclerotic vessels showed similar numbers of platelets in both phenotypes. However, the attached platelets in bleeder pigs showed significantly less spreading than did those in the normal animals. The results show that injury to intimal foam cell and mixed cellular lesions in coronary arteries of cholesterol-fed swine does not promote the development of platelet-fibrin thrombus formation. In contrast, when the injury extended to the media, mixed thrombi were formed.

A review was undertaken of the clinical, gross, and microscopic features of thyroid carcinoma in all patients younger than 21 years of age seen at North Carolina Memorial Hospital from 1952 to 1987 (N = 32). These patients had papillary carcinoma, well-differentiated follicular carcinoma with Hurthle cell change, medullary carcinoma, and an unclassifiable aggressive malignancy. In spite of the presence of lymph node metastases at diagnosis in more half the patients with papillary carcinoma, the prognosis of pediatric papillary thyroid carcinoma appears to be excellent with treatment by surgical debulking and hormonal thyroid suppression. Flow cytometric study of 26 cases showed tumor aneuploidy in 8 of 21 papillary carcinomas and 2 of 3 follicular carcinomas. Aneuploidy did not, however, correlate with clinical outcome in this group of pediatric patients, who were followed for 1 to 29 years. Analysis of multiple tissue blocks of tumor does appear to increase the probability of identifying aneuploid populations.

The clinical course of a 17-year-old boy with primary osteogenic sarcoma of the left atrium with partial obstruction of the mitral valve and the right pulmonary veins is described. After operative removal of the tumor, echocardiography documented its rapid recurrence. Despite two subsequent open-heart operations and adjuvant chemotherapy and radiotherapy, the patient died twenty-one months after the initial symptoms. Previous reports of such tumors are reviewed, and technical difficulties of removal are discussed.

This study was designed to examine the functional characteristics of thyroid adenomas, adenomatous (colloid) nodules, hyperfunctioning nodules, and normal thyroid parenchyma with respect to the presence of TSH receptors, levels of adenylate cyclase, and the responsiveness of adenylate cyclase to specific stimuli, e.g., NaF (sodium fluoride), GTPγS [guanosine 5′-0-(3-thiotriphosphatate)], PGE1, and TSH. Aliquots of specimens from patients undergoing thyroidectomy [11 normal thyroids, 7 adenomas, 8 adenomatous (colloid) nodules, 5 hyperfunctioning nodules] were immersed in liquid nitrogen and stored at 90°C. Studies were performed on partially purified plasma membranes. Receptor binding of125I-TSH was quantitated and analyzed according to Scatchard. Adenylate cyclase activity was assayed in the same tissue fraction by conversion of (α-32P) ATP to cAMP.

The early response to coronary artery injury was investigated in normal swine and in swine with von Willebrand disease (vWD). Thirty minutes after coronary endothelial denudation, a monolayer of platelets was adherent to areas of simple injury in both bleeder and normal swine. The number of adherent platelets was not significantly different in the two phenotypes. Injury involving the media of the vessel produced platelet-fibrin thrombi. Platelet activation, as judged by pseudopod formation and platelet spreading over areas of simple injury, was significantly less in bleeder animals than in normal animals. These studies suggest that chemotaxis and initial contact adhesion of platelets to injured arterial wall is independent of the von Willebrand factor. On the other hand, the spreading and activation of platelets on the subendothelium appear to be dependent on the presence of plasma von Willebrand factor. Through this mechanism von Willebrand factor may contribute to arterial thrombosis and atherogenesis.

A 33-year-old woman presenting with secondary amenorrhea and galactorrhea was found to have a Sertoli cell tumor of the ovary. The neoplasm also had a sex cord tumor with annular tubules (SCTAT) component. Further investigations revealed that in many respects the patient was endocrinologically pregnant. She had markedly elevated serum estrogen and progesterone levels and the endometrium demonstrated pronounced decidualization, but there was no evidence of actual pregnancy. Estrogen and progesterone were demonstrated by immunohistochemistry to be present in both the Sertoli cell and SCTAT portions of the tumor.

Ultrastructural examination of 26 osteogenic sarcomas has revealed that in addition to the basic osteoblast comprising the tumor, five additional cell types may be identified. These include chondroblasts, osteocytes, undifferentiated cells and myofibroblasts. The latter cell type has not been reported in previous studies of medullary osteosarcomas. The relative portions of the cell types varied as to the type of osteogenic sarcoma studied. Osteoid production, the single diagnostic feature of these tumors, was evident and the various ultrastructural appearances of this material are shown. One case of an osteogenic sarcoma that arose in Paget's disease of bone is described and did not differ ultrastructurally from the conventional medullary osteogenic sarcoma. Our studies have shown that osteogenic sarcoma may share common ultrastructural features with other sarcomas involving bone and soft tissue and that the basic difference between osteogenic sarcoma and soft-tissue sarcomas is the presence of osteoid in the former and differing cytoplasmic differentiation in the latter category of tumors.