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Supplementary Figure 3 from Immune Competency of a Hairless Mouse Strain for Improved Preclinical Studies in Genetically Engineered Mice

Supplementary Fig. 1. IB inhibits the growth of breast cancer cells in a dose and time dependent manner. Supplementary Fig. 2. IB inhibits migration and invasion of breast cancer cells. Supplementary Fig. 3. IB treatment inhibits in vivo growth and metastasis of breast tumorigenesis. Supplementary Fig. 4. IB is safe and effective against human breast cancers. Supplementary Fig. 5. (A) Western blot analysis of MCF-7 cells treated with vehicle or IB (5μM) using antibodies against FOXM1, SKP2, PLK1 and XRCC3. Supplementary Fig. 6. (A) Western blot analysis of MCF-7 cells treated with vehicle or IB (5μM) using antibodies against cyclin D 1, CDK4, cyclin E2, CDK2. Supplementary Fig. 7. (A) A549, HeLa and DU145 cells were treated with various doses of IB (1-5 μM) for 24 hours and then cells were re-seeded in 6-well plate and allowed to grow for additional 7 days before colonies were stained with crystal violet.

Supplementary Figure 1 from Immune Competency of a Hairless Mouse Strain for Improved Preclinical Studies in Genetically Engineered Mice

Supplementary Figure 2 from Immune Competency of a Hairless Mouse Strain for Improved Preclinical Studies in Genetically Engineered Mice

Purpose: The approaches aimed at inhibiting the ability of cancer cells to repair DNA strand breaks have emerged as promising targets for treating cancers. Here, we assessed the potential of imipramine blue (IB), a novel analogue of antidepressant imipramine, to suppress breast cancer growth and metastasis by inhibiting the ability of breast cancer cells to repair DNA strand breaks by homologous recombination (HR).Experimental Design: The effect of IB on breast cancer growth and metastasis was assessed in vitro as well as in preclinical mouse models. Besides, the therapeutic efficacy and safety of IB was determined in ex vivo explants from breast cancer patients. The mechanism of action of IB was evaluated by performing gene-expression, drug–protein interaction, cell-cycle, and DNA repair studies.Results: We show that the systemic delivery of IB using nanoparticle-based delivery approach suppressed breast cancer growth and metastasis without inducing toxicity in preclinical mouse models. Using ex vivo explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cells. Furthermore, our mechanistic studies revealed that IB may interact and inhibit the activity of proto-oncogene FoxM1 and associated signaling that play critical roles in HR-mediated DNA repair.Conclusions: These findings highlight the potential of IB to be applied as a safe regimen for treating breast cancer patients. Given that FoxM1 is an established therapeutic target for several cancers, the identification of a compound that inhibits FoxM1- and FoxM1-mediated DNA repair has immense translational potential for treating many aggressive cancers. Clin Cancer Res; 22(14); 3524–36. ©2016 AACR.

Genetically engineered mouse models (GEMM) of cancer are of increasing value to preclinical therapeutics. Optical imaging is a cost-effective method of assessing deep-seated tumor growth in GEMMs whose tumors can be encoded to express luminescent or fluorescent reporters, although reporter signal attenuation would be improved if animals were fur-free. In this study, we sought to determine whether hereditable furlessness resulting from a hypomorphic mutation in the Hairless gene would or would not also affect immune competence. By assessing humoral and cellular immunity of the SKH1 mouse line bearing the hypomorphic Hairless mutation, we determined that blood counts, immunoglobulin levels, and CD4+ and CD8+ T cells were comparable between SKH1 and the C57Bl/6 strain. On examination of T-cell subsets, statistically significant differences in naïve T cells (1.7 versus 3.4 × 105 cells/spleen in SKH1 versus C57Bl/6, P = 0.008) and memory T cells (1.4 versus 0.13 × 106 cells/spleen in SKH1 versus C57Bl/6, P = 0.008) were detected. However, the numerical differences did not result in altered T-cell functional response to antigen rechallenge (keyhole limpet hemocyanin) in a lymph node cell in vitro proliferative assay. Furthermore, interbreeding the SKH1 mouse line to a rhabdomyosarcoma GEMM showed preserved antitumor responses of CD56+ natural killer cells and CD163+ macrophages, without any differences in tumor pathology. The fur-free GEMM was also especially amenable to multiplex optical imaging. Thus, SKH1 represents an immune competent, fur-free mouse strain that may be of use for interbreeding to other genetically engineered mouse models of cancer for improved preclinical studies. Mol Cancer Ther; 9(8); 2354–64. ©2010 AACR.

Supplimentary Methods and Figure Legends

Supplementary Table S1: Primers used in the present investigation. Supplementary Table S 2 : Gene Ontology Enrichment Analysis on differentially expressed genes in IB-treated compared to vehicle-treated breast cancer cells using Database for Annotation, Visualization and Integrated Discovery (DAVID). BP- Biological Processes.

Hepatocellular carcinoma (HCC) is one of the most common and deadly human cancers and it remains poorly managed. Human HCC development is often associated both with elevated expression of inducible nitric oxide synthase (iNOS) and with genetic deletion of the major denitrosylase S-nitrosoglutathione reductase (GSNOR/ADH5). However, their causal involvement in human HCC is not established. In mice, GSNOR deficiency causes S-nitrosylation and depletion of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT) and increases rates of both spontaneous and DEN carcinogen-induced HCC. Here, we report that administration of 1400W, a potent and highly selective inhibitor of iNOS, blocked AGT depletion and rescued the repair of mutagenic O6-ethyldeoxyguanosines following DEN challenge in livers of GSNOR-deficient (GSNOR−/−) mice. Notably, short-term iNOS inhibition following DEN treatment had little effect on carcinogenesis in wild-type mice, but was sufficient to reduce HCC multiplicity, maximal size, and burden in GSNOR−/− mice to levels comparable with wild-type controls. Furthermore, increased HCC susceptibility in GSNOR−/− mice was not associated with an increase in interleukin 6, tumor necrosis factor-α, oxidative stress, or hepatocellular proliferation. These results suggested that GSNOR deficiency linked to defective DNA damage repair likely acts at the tumor initiation stage to promote HCC carcinogenesis. Together, our findings provide the first proof of principle that HCC development in the context of uncontrolled nitrosative stress can be blocked by pharmacologic inhibition of iNOS, possibly providing an effective therapy for patients with HCC. Cancer Res; 73(9); 2897–904. ©2013 AACR.

PDF File - 91K, Lack of effect of 1400W on HCC cell growth.

Purpose: The approaches aimed at inhibiting the ability of cancer cells to repair DNA strand breaks have emerged as promising targets for treating cancers. Here, we assessed the potential of imipramine blue (IB), a novel analogue of antidepressant imipramine, to suppress breast cancer growth and metastasis by inhibiting the ability of breast cancer cells to repair DNA strand breaks by homologous recombination (HR). Experimental Design: The effect of IB on breast cancer growth and metastasis was assessed in vitro as well as in preclinical mouse models. Besides, the therapeutic efficacy and safety of IB was determined in ex vivo explants from breast cancer patients. The mechanism of action of IB was evaluated by performing gene-expression, drug–protein interaction, cell-cycle, and DNA repair studies. Results: We show that the systemic delivery of IB using nanoparticle-based delivery approach suppressed breast cancer growth and metastasis without inducing toxicity in preclinical mouse models. Using ex vivo explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cells. Furthermore, our mechanistic studies revealed that IB may interact and inhibit the activity of proto-oncogene FoxM1 and associated signaling that play critical roles in HR-mediated DNA repair. Conclusions: These findings highlight the potential of IB to be applied as a safe regimen for treating breast cancer patients. Given that FoxM1 is an established therapeutic target for several cancers, the identification of a compound that inhibits FoxM1- and FoxM1-mediated DNA repair has immense translational potential for treating many aggressive cancers. Clin Cancer Res; 22(14); 3524–36. ©2016 AACR.

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.

In women, age-related bone loss is associated with increased risk of bone fracture. Existing therapies are associated with severe side effects; thus, there is a need to find alternative medicines with less or optimal side effects. Cissus quadrangularis (CQ), an Ayurvedic medicine used to enhance fracture healing, was tested for its bone protective properties and studied to discern the mechanism by which it is beneficial to bone. Female Sprague Dawley rats were either sham operated or ovariectomized and were fed CQ for 3 months. Several biochemical markers, cytokines and hormones were assayed. Femur, tibia and lumbar vertebrae were subjected to pQCT and µCT densitometry. MC3T3 cells were cultured, treated with CQ and used to analyze miRNA content and subjected to qPCR for gene expression analysis related to bone metabolism. CQO rats showed protected bone mass and microarchitecture of trabecular bone in the distal femoral metaphysis and the proximal tibial metaphysis. The lumbar vertebrae, however, showed no significant changes. Serum protein expression levels of P1NP increased and Trap5b and CTX levels decreased with in vivo CQ treatment. Some influence on the anti- and pro-inflammatory markers was also observed. Significantly high level of estradiol in the CQO rats was observed. In vitro expression of a few genes related to bone metabolism showed that osteocalcin increased significantly. The other genes—collagen I expression, SPP1, BMP2, DCAT1—decreased significantly. Certain miRNA that regulate bone turnover using the BMP pathway and Wnt signaling pathways were upregulated by CQ. qPCR after acute treatment with CQ showed significantly increased levels of osteocalcin and decreased levels of Wnt/β catenin antagonist DCAT1. Overall, CQ protected the microarchitecture of the long bones from ovariectomy-induced bone loss. This may be because of decreased inflammation and modulation through the BMP and Wnt signaling pathways. We conclude that CQ is a potential therapeutic agent to treat postmenopausal osteoporosis with no side effects.

The baboon provides a natural non-human primate model for photosensitive, generalized epilepsy. This study describes an implantation procedure for the placement of subdural grid and strip electrodes for continuous video-EEG monitoring in the epileptic baboon to evaluate the generation and propagation of ictal and interictal epileptic discharges. Subdural grid, strip and depth electrodes were implanted in six baboons, targeting brain regions that were activated in functional neuroimaging studies during photoparoxysmal responses. The baboons were monitored with continuous video-EEG monitoring for 2-21 (mean 9) days. Although the animals were tethered, the EEG signal was transmitted wirelessly to optimize their mobility. Spontaneous seizures, interictal epileptic discharges (IEDs), and responses to intermittent light stimulation (ILS) were assessed. Due to cortical injuries related to the electrode implantation and their displacement, the procedure was modified. Habitual myoclonic and generalized tonic-clonic seizures were recorded in three baboons, all associated with a generalized ictal discharge, but were triggered multiregionally, in the frontal, parietal and occipital cortices. IEDs were similarly expressed multiregionally, and responsible for triggering most generalized spike-and-wave discharges. Generalized photoparoxysmal responses were activated only in one baboon, while driving responses recorded in all three photosensitive baboons were 2.5 times the stimulus rate. In contrast to previous intracranial investigations in this model, generalized ictal and interictal epileptic discharges were triggered by parietal and occipital, in addition to the frontocentral cortices. Furthermore, targeted visual areas responded differently to ILS in photosensitive than nonphotosensitive baboons, but further studies are required before mechanisms can be implicated for ILS-induced activation of the epileptic networks.

Overexpression of platelet-derived growth factor receptor alpha (PDGFR-A) has been documented in association with primary tumors and metastasis in medulloblastoma. Tumors from our genetically engineered sonic hedgehog-driven medulloblastoma mouse model overexpress PDGFR-A in primary tumors and thus this mouse model is a good platform with which to study the role of PDGFR-A in this central nervous system malignancy. We hypothesized that inhibition of PDGFR-A in medulloblastoma can slow or inhibit tumor progression in living individuals. To test our hypothesis, we targeted PDGFR-A mediated tumor growth in vitro and in vivo using the tyrosine kinase inhibitor, tandutinib (MLN-518), which strongly inhibits PDGFR-A. Although PDGFR-A inhibition by this agent resulted in reduced mouse tumor cell growth and increased apoptosis in vitro, and reduced tumor cell proliferation in vivo, tandutinib did reduce tumor volume at the doses tested (360 mg/kg) in vivo. Thus, tandutinib may be an agent of interest for sonic hedgehog-driven medulloblastoma if a synergistic drug combination can be identified.

Naked mole rats (NMRs; Heterocephalus glaber) are the longest-living rodents known, with a maximum lifespan of 30 years--5 times longer than expected on the basis of body size. These highly social mouse-sized rodents, naturally found in subterranean burrows in the arid and semiarid regions of the horn of Africa, are commonly used in behavioral, neurological, and ecophysiological research. Very old NMRs (28 years), like humans, show signs of age-associated pathologies (e.g., muscle loss) as well as the accumulation of lipofuscin pigments, but no signs of tumorigenesis. Indeed, for at least 80% of their lives NMRs maintain normal activity, body composition, and reproductive and physiological functions with no obvious age-related increases in morbidity or mortality rate. Their long lifespan is attributed to sustained good health and pronounced cancer resistance. Clearly physiological and biochemical processes in this species have evolved to dramatically extend both their good health- and lifespan. We and others have tested various current theories using this species as an exceptionally long-lived animal model of successful abrogated aging. Surprisingly, NMRs have high levels of oxidative stress and relatively short telomeres, yet they are extremely resilient when subjected to cellular stressors and appear capable of sustaining both their genomic and protein integrity under hostile conditions. The challenge is to understand how these animals are able to do this. Elucidating these mechanisms will provide useful information for enhancing human life- and healthspan, making the naked mole rat a true "supermodel" for aging research and resistance to chronic age-associated diseases.

Genetically engineered mouse models (GEMM) of cancer are of increasing value to preclinical therapeutics. Optical imaging is a cost-effective method of assessing deep-seated tumor growth in GEMMs whose tumors can be encoded to express luminescent or fluorescent reporters, although reporter signal attenuation would be improved if animals were fur-free. In this study, we sought to determine whether hereditable furlessness resulting from a hypomorphic mutation in the Hairless gene would or would not also affect immune competence. By assessing humoral and cellular immunity of the SKH1 mouse line bearing the hypomorphic Hairless mutation, we determined that blood counts, immunoglobulin levels, and CD4+ and CD8+ T cells were comparable between SKH1 and the C57Bl/6 strain. On examination of T-cell subsets, statistically significant differences in naïve T cells (1.7 versus 3.4 × 105 cells/spleen in SKH1 versus C57Bl/6, P = 0.008) and memory T cells (1.4 versus 0.13 × 106 cells/spleen in SKH1 versus C57Bl/6, P = 0.008) were detected. However, the numerical differences did not result in altered T-cell functional response to antigen rechallenge (keyhole limpet hemocyanin) in a lymph node cell in vitro proliferative assay. Furthermore, interbreeding the SKH1 mouse line to a rhabdomyosarcoma GEMM showed preserved antitumor responses of CD56+ natural killer cells and CD163+ macrophages, without any differences in tumor pathology. The fur-free GEMM was also especially amenable to multiplex optical imaging. Thus, SKH1 represents an immune competent, fur-free mouse strain that may be of use for interbreeding to other genetically engineered mouse models of cancer for improved preclinical studies. Mol Cancer Ther; 9(8); 2354–64. ©2010 AACR.

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.

One way to better understand the contribution of DNA repair, DNA damage, and mutagenesis in aging would be to enhance DNA repair activity, lower DNA damage, and lower mutagenesis. Because the repair protein O6-methylguanine-DNA methyltransferase (MGMT) acts alone and stoichiometrically, the human MGMT (hMGMT) cDNA was selected to test the feasibility of enhancing DNA repair activity in transgenic mice. MGMT activity is largely responsible for ameliorating the deleterious effects of O6-methylguanine (O6mG) lesions in DNA in a direct reversal mechanism. A transgene was constructed consisting of a portion of the human transferrin (TF) promoter and hMGMT cDNA such that hMGMT is expressed in transgenic mouse brain and liver. Expression of hMGMT was associated with a significant reduction in the occurrence of an age-related hepatocellular carcinoma in male mice at 15 months of age. Longitudinal and cross-sectional studies were initiated to determine whether the reduced incidence of hepatocellular carcinoma would impact median or maximum life span. The cross-sectional study performed on 15-month-old male animals confirmed the reduced occurrence of spontaneous hepatocellular carcinoma. At 30 months of age, however, the occurrence of hepatocellular carcinoma in at least one transgenic line was similar to that for nontransgenic animals. The longitudinal study is ongoing; however, at present no significant differences in life span have been detected. Tissues expressing the MGMT transgene also displayed greater resistance to alkylation-induced tumor formation. These results suggest that transgenes can be used to direct enhanced DNA repair gene expression and that enhanced expression can protect animals from certain spontaneous and induced tumors.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal-dominant disorder characterized by the age-dependent development of focal arteriovenous malformations and telangiectases. HHT type 2 is caused by loss of function mutations in activin receptor-like kinase 1 (ACVRL 1 or ALK1). However, the factors that initiate lesion formation and those that influence disease progression remain unknown. Because heterozygous mice contain the appropriate genotype for an animal model of this disorder, mice heterozygous for a loss-of-function mutation in Acvrll were carefully examined for an HHT-like phenotype. These mice developed age-dependent vascular lesions in the skin, extremities, oral cavity and in the internal organs (lung, liver, intestine, spleen and brain), similar to those seen in HHT patients. Major histopathological features of the lesions included thin-walled dilated vessels in close proximity to each other, hemorrhage and fibrosis. Similar to HHT patients, the mice also exhibited gastrointestinal bleeding, as evidenced by positive fecal occult blood tests. An Acvrl1 +/- mouse with profound liver involvement also displayed a secondary cardiac phenotype, similar to that observed in human patients. The similarity of affected organs, age-dependent penetrance, histological similarity of the lesions and recapitulation of a secondary phenotype suggest that the Acvrl1 +/- mice are an appropriate animal model for the identification of additional genetic and environmental factors that cause pathology in HHT type 2 patients. In addition, studies utilizing this animal model can yield valuable information on the role of ALK1 in maintenance of adult vascular architecture including arteriovenous identity.

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.

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

Hepatocellular carcinoma (HCC) is one of the most common and deadly human cancers and it remains poorly managed. Human HCC development is often associated both with elevated expression of inducible nitric oxide synthase (iNOS) and with genetic deletion of the major denitrosylase S-nitrosoglutathione reductase (GSNOR/ADH5). However, their causal involvement in human HCC is not established. In mice, GSNOR deficiency causes S-nitrosylation and depletion of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT) and increases rates of both spontaneous and DEN carcinogen-induced HCC. Here, we report that administration of 1400W, a potent and highly selective inhibitor of iNOS, blocked AGT depletion and rescued the repair of mutagenic O6-ethyldeoxyguanosines following DEN challenge in livers of GSNOR-deficient (GSNOR−/−) mice. Notably, short-term iNOS inhibition following DEN treatment had little effect on carcinogenesis in wild-type mice, but was sufficient to reduce HCC multiplicity, maximal size, and burden in GSNOR−/− mice to levels comparable with wild-type controls. Furthermore, increased HCC susceptibility in GSNOR−/− mice was not associated with an increase in interleukin 6, tumor necrosis factor-α, oxidative stress, or hepatocellular proliferation. These results suggested that GSNOR deficiency linked to defective DNA damage repair likely acts at the tumor initiation stage to promote HCC carcinogenesis. Together, our findings provide the first proof of principle that HCC development in the context of uncontrolled nitrosative stress can be blocked by pharmacologic inhibition of iNOS, possibly providing an effective therapy for patients with HCC. Cancer Res; 73(9); 2897–904. ©2013 AACR.

Publisher Summary Although guinea pigs are sensitive and susceptible to the development of lesions from a wide range of viruses, bacteria, protozoa, and parasites, only a small number of organisms cause natural infection and only a portion of that group cause clinical disease. This chapter discusses naturally occurring diseases of guinea pigs, although some data from experimental infections have also been covered as they relate to the pathogenesis of the disease. The material presented includes background, etiology, epizootiology/pathogenesis, clinical manifestations, pathology, diagnosis, prevention, and therapy. The diseases are discussed in an alphabetical order based on the taxonomic groups to which the organisms belong and are independent of the order of perceived importance of the various diseases. The Federation of European Laboratory Animal Science Associations recommends monitoring for guinea pig adenovirus, guinea pig cytomegalovirus, Sendai virus, ectoparasites, endoparasites, E. cunniculi, and a variety of bacteria including Bordetella bronchiseptica, Chlamydia psittaci, Corynebacterium kutscheri, dermatophytes, Pasteurellaceae, Salmonella, Streptobacillus moniliformis, Streptococcus, Yersinia pseudotuberculosis, and Clostridium piliforme. Virus-associated necrotizing ronchopneumonia in guinea pigs is a spontaneous multifactorial disease that has low morbidity, high mortality, and a worldwide distribution.

Publisher Summary Naked mole rats are mouse-sized rodents that have become an important animal model in biomedical research. They play a unique mammalian role in behavioral and ecophysiological research of life underground. This chapter studies the general physiology, anatomy of organ systems, husbandry, and uses in research of the naked mole rats. Naked mole rats belong to the order Rodentia in that they have two incisor teeth on the upper and lower arcade that continuously grow. The skin is loose, wrinkled, and brownish pink in color. The body is for the most part absent of hairs with the exception of tactile hairs that are regularly arranged throughout the body and which are particularly prominent around the face and to a lesser extent on the tail. They are typically housed at 28–30°C, and at 50–60% relative humidity. Because naked mole rats are social and have cooperative behaviors, the study of their conduct has more applicability to people. The chapter describes the models of experimental research on the naked mole rat such as the model of reproductive suppression, model of somatosensory processing, model of bone elongation, and model of aging.

Background Laparoscopy, which is a minimally invasive surgery, is associated with decreased peritoneal adhesions and inflammatory response compared with laparotomy. Objective To evaluate whether natural orifice transluminal endoscopic surgery (NOTES) leads to an attenuated peritoneal response compared with laparoscopy. Design Pooled histologic analysis from 2 randomized porcine trials. Setting Laboratory. Intervention Histologic analysis of swine undergoing diagnostic laparoscopy, diagnostic NOTES peritoneoscopy, NOTES with transgastric mesh placement, or diagnostic endoscopy (no gastrotomy) followed by laparoscopic mesh placement. Main Outcome Measurements The presence and grade of inflammation in necropsy specimens of lung, liver, and spleen as reviewed by a blinded veterinary pathologist. Results Four NOTES mesh animals exhibited mesh infections at necropsy. Tissue from 48 swine were available for analysis. Pulmonary inflammation, liver fibrosis, and spleen capsulitis were the primary findings. No difference was seen in the incidence of each finding among groups. The severity of the pulmonary inflammation in the laparoscopy group was significantly higher than in the NOTES groups. The NOTES mesh group exhibited significantly more severe liver fibrosis and spleen capsulitis. There was no difference between clinical behavior, serum white blood cell count, or peritoneal white blood cell count among groups in either study. Intra-abdominal pressures during NOTES were lower than during laparoscopy. Limitations Pooled analysis of 2 separate studies. Conclusion More severe pulmonary inflammation was found in animals undergoing longer laparoscopic procedures with higher intra-abdominal pressures. Intraperitoneal inflammation was most significant with transgastric mesh placement, likely caused by infections.

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV ("preS2 mutant") that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged--2-year-old--mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines--expressing either mutant or wildtype HBV--therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC.

Human hepatocellular carcinoma (HCC) is associated with elevated expression of inducible nitric oxide synthase (iNOS), but the role of nitric oxide in the pathogenesis of HCC remains unknown. We found that the abundance and activity of S -nitrosoglutathione reductase (GSNOR), a protein critical for control of protein S -nitrosylation, were significantly decreased in ~50% of patients with HCC. GSNOR-deficient mice were very susceptible to spontaneous and carcinogen-induced HCC. During inflammatory responses, the livers of GSNOR-deficient mice exhibited substantial S -nitrosylation and proteasomal degradation of the key DNA repair protein O 6 -alkylguanine-DNA alkyltransferase. As a result, repair of carcinogenic O 6 -alkylguanines in GSNOR-deficient mice was significantly impaired. Predisposition to HCC, S -nitrosylation and depletion of alkylguanine-DNA alkyltransferase, and accumulation of O 6 -alkylguanines were all abolished in mice deficient in both GSNOR and iNOS. Thus, our data suggest that GSNOR deficiency, through dysregulated S -nitrosylation, may promote HCC, possibly by inactivating a DNA repair system.

Publisher Summary The congenitally athymic nude rat (rnu/rnu) is a genetic mutant found in Rattus norvegicus characterized by a severe alteration in the cell-mediated branch of immunity (CMI). Altered cellular immune responses are owing to a lack of appropriate development and education of functional T-cell (thymus-derived) lymphocytes. The size and robustness of the nude rat and the absence of major endocrinological abnormalities make it an appropriate experimental model for a variety of immunological, surgical, infectious, transplant-related, and oncological procedures. Nude rats are uniquely capable of handling an increased tumor burden without overt distress, and the additional body size allows therapeutic intervention more easily than in a nude mouse. Therapeutic data for anti-neoplastic studies are presented in terms of (1) increased lifespan reflected by the relative median survival time of treated verses control groups (%treated/control); (2) cures; or (3) relative median times (in days) for the treated and control groups to grow tumors of a predetermined target size. The nude rat model has demonstrated value for many areas of biomedical research. From basic cellular function of immune cells to intricate interactions of tumors, treatment, and imaging modalities, the nude rat has provided a robust model of suitable size and appropriate response.

Hospitalization of the elderly for invasive pneumococcal disease is frequently accompanied by the occurrence of an adverse cardiac event; these are primarily new or worsened heart failure and cardiac arrhythmia. Herein, we describe previously unrecognized microscopic lesions (microlesions) formed within the myocardium of mice, rhesus macaques, and humans during bacteremic Streptococcus pneumoniae infection. In mice, invasive pneumococcal disease (IPD) severity correlated with levels of serum troponin, a marker for cardiac damage, the development of aberrant cardiac electrophysiology, and the number and size of cardiac microlesions. Microlesions were prominent in the ventricles, vacuolar in appearance with extracellular pneumococci, and remarkable due to the absence of infiltrating immune cells. The pore-forming toxin pneumolysin was required for microlesion formation but Interleukin-1β was not detected at the microlesion site ruling out pneumolysin-mediated pyroptosis as a cause of cell death. Antibiotic treatment resulted in maturing of the lesions over one week with robust immune cell infiltration and collagen deposition suggestive of long-term cardiac scarring. Bacterial translocation into the heart tissue required the pneumococcal adhesin CbpA and the host ligands Laminin receptor (LR) and Platelet-activating factor receptor. Immunization of mice with a fusion construct of CbpA or the LR binding domain of CbpA with the pneumolysin toxoid L460D protected against microlesion formation. We conclude that microlesion formation may contribute to the acute and long-term adverse cardiac events seen in humans with IPD., Author Summary Hospitalization for community-acquired pneumonia carries a documented risk for adverse cardiac events. These occur during infection and contribute to elevated mortality rates in convalescent individuals up to 1 year thereafter. We describe a previously unrecognized pathogenic mechanism by which Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, causes direct cardiotoxicity and forms microscopic bacteria-filled lesions within the heart. Microlesions were detected in experimentally infected mice and rhesus macaques, as well as in heart sections from humans who succumbed to invasive pneumococcal disease (IPD). Cardiac microlesion formation required interaction of the bacterial adhesin CbpA with host Laminin receptor and bacterial cell wall with Platelet-activating factor receptor. Microlesion formation also required the pore-forming toxin pneumolysin. When infected mice were rescued with antibiotics, we observed robust signs of collagen deposition at former lesion sites. Thus, microlesions and the scarring that occurs thereafter may explain why adverse cardiac events occur during and following IPD.

Bile is an important physiological bodily fluid which functions in the regulation of cholesterol metabolism, promotes the absorption of lipid and fat-soluble vitamins by the gut and serves in the excretion of toxic substances from the liver. Conversely, due to autooxidative processes bile is highly toxic to the hepatocyte and gastrointestinal epithelium. In this investigation, extremely high day time physiological levels of the endogenous antioxidant, melatonin, were measured in the bile of several mammals including rat, guinea pig, rabbit, pig, monkey and humans. Melatonin concentrations in the bile samples ranged from 2,000 to 11,000 pg/ml when measured by radioimmunoassay (RIA). These melatonin levels in bile are 2 to 3 orders of magnitude higher than those in day time serum. The presence of melatonin in bile was confirmed by HPLC with an electrochemical detector. This method, like the RIA, also documented very high levels of melatonin in bile. The presence of high levels of melatonin in bile may be essential to prevent oxidative damage to biliary and small intestinal epithelium induced by bile acids and oxidized cholesterol derivatives.

Botulism is caused by intoxication with one or more of the seven neurotoxins produced by Clostridium botulinum. Food poisoning is the most common cause of botulism. Accidental inhalation exposure, however, has been reported in the laboratory,4 and inhalation is the likely route of exposure after the toxin’s use as a terrorist weapon or warfare agent. Toxoids are available for immunization, but there are presently no known drugs that can be used to prevent or treat botulinum intoxication. A pentavalent human, hyperimmune globulin product6 and a heptavalent, equine F(ab’)2 (despeciated) antitoxin (unpublished data, G.E. Lewis, Jr. and R.M. Condie) were evaluated in a preliminary study as prophylaxis or therapy for inhalation botulism (serotype A) in rhesus monkeys.

African filoviruses have caused outbreaks of fulminating hemorrhagic fever among humans. In 1989, related filoviruses were isolated from cynomolgus monkeys imported into the United States from the Philippines. The pathogenic potential of these new filoviruses was compared in 16 Asian monkeys (Macaca fascicularis-cynomolgus) and 16 African monkeys (Cercopithecus aethiops-African green) using African filoviruses from Zaire (Ebola virus) and Sudan or Asian filoviruses (Reston and Pennsylvania). African filovirus infections resulted in earlier death (P = .005), had a shorter duration of disease and median incubation period (3-4 vs. 7 days), and had earlier peak viremia (5-7 vs. 7-9 days). African green monkeys showed significantly higher survival than cynomolgus monkeys (P less than .01), and some were asymptomatic as have been humans accidentally infected with Asian filovirus. Rechallenge experiments showed that protection in survivors of filovirus infections against fatal challenge with Ebola (Zaire) virus is unpredictable. The minimal clinical disease observed in humans infected with the Reston strain is consistent with host- and virus-dependent pathogenicity.

RESULTS: IL-18Bpa was expressed and secreted by the prostate cancer cell lines DU145 and PC3, but not by LNCaP and CWR22, upon interferon(IFN) stimulation. IFN-induced secretion of IL-18Bpa was enhanced by added TNF, IFNand IFN. The IL-18Bpa secreted from DU145 and PC3 functionally inhibited IL-18. Conditioned medium from IL-18Bpa-overexpressed PC3 cells suppressed CD8+ IFN+ cells and CD4+ cells and appeared to decrease TH1 cells in human peripheral blood cultures. Immunohistochemical analyses showed positive IL18Bpa staining in prostate cancer cells as well as in macrophages in radical prostatectomy specimens. Significant differences in post-DRE urinary IL-18Bpa levels (normalized by total protein) were found between cases with and without cancer on biopsy (P = 0.02) and serum IL-18Bpa levels correlated with Gleason score (P = 0.03). CONCLUSIONS: Our finding of elevated IL-18Bpa secretion from prostate cancer cells suggests an attempt by cancer to escape immune surveillance. IL-18Bpa merits further study as a marker of aggressive prostate cancer and as a therapeutic target.

The acute intramuscular toxicity of MMB-4 was determined in male ICR mice by using the single dose method. LD(1), LD(50), and LD(95) with their 95% confidence limits were calculated by probit analysis. The LD(50) was 448 mg/kg with the 95% confidence limit (398 mg/kg, 506 mg/kg). The MMB-4 formulation falls in the very toxic range.