Epidemiological and molecular characterization of a novel adenovirus of squirrel monkeys after fatal infection during immunosuppression.

Adenoviruses are a frequent cause of acute upper respiratory tract infections that can also cause disseminated disease in immunosuppressed patients. We identified a novel adenovirus, squirrel monkey adenovirus 1 (SqMAdV-1), as the cause of fatal infection in an immunocompromised squirrel monkey (Saimiri boliviensis) at the Keeling Center for Comparative Medicine and Research (KCCMR). Sequencing of SqMAdV-1 revealed that it is most closely related (80.4 % pairwise nucleotide identity) to the titi monkey (Plecturocebus cupreus) adenovirus (TMAdV). Although identified in the titi monkey, TMAdV is highly lethal in these monkeys, and they are not thought to be the natural host. While SqMAdV-1 is similar to other primate adenoviruses in size and genomic characteristics, a nucleotide polymorphism at the expected stop codon of the DNA polymerase gene results in a 126 amino acid extension at the carboxy terminus, a feature not previously observed among other primate adenoviruses. PCR testing and partial sequencing of 95 archived faecal samples from other squirrel monkeys (Saimiri boliviensis and Saimiri sciureus) housed at the KCCMR revealed the presence of three distinct, and apparently endemic species of adenoviruses. A grouping of ten squirrel monkey adenovirus variants has high similarity to SqMAdV-1. A single adenovirus variant (designated SqMAdV-3), detected in five monkeys, has similarity to tufted capuchin (Sapajus apella) adenoviruses. The largest group of adenovirus variants detected (designated SqMAdV-2.0-2.16) has very high similarity (93-99 %) to the TMAdV, suggesting that squirrel monkeys may be the natural host of the TMAdV.

Experimental Zika Virus Infection of Neotropical Primates.

The establishment of a sylvatic reservoir of Zika virus (ZIKV) in the Americas is dependent on the susceptibility of primates of sufficient population density, the duration and magnitude of viremia, and their exposure to the human mosquito-borne transmission cycle. To assess the susceptibility of squirrel (Saimiri sp.) and owl monkeys (Aotus sp.) to infection, we inoculated four animals of each species with ZIKV from the current epidemic. Viremia in the absence of detectible disease was observed in both species and seroconversion occurred by day 28. ZIKV was detected in the spleen of three owl monkeys: one at 7 days postinoculation (dpi) and two at 14 dpi. This study confirms the susceptibility to ZIKV infection of two Neotropical primate species that live in close proximity to humans in South America, suggesting that they could support a widespread sylvatic ZIKV cycle there. Collectively, establishment of a ZIKV sylvatic transmission cycle in South America would imperil eradication efforts and could provide a mechanism for continued exposure of humans to ZIKV infection and disease.

Endemic Viruses of Squirrel Monkeys (Saimiri spp.).

Nonhuman primates are the experimental animals of choice for the study of many human diseases. As such, it is important to understand that endemic viruses of primates can potentially affect the design, methods, and results of biomedical studies designed to model human disease. Here we review the viruses known to be endemic in squirrel monkeys (Saimiri spp.). The pathogenic potential of these viruses in squirrel monkeys that undergo experimental manipulation remains largely unexplored but may have implications regarding the use of squirrel monkeys in biomedical research.

Supporting a role for the GTPase Rab7 in prostate cancer progression.

Invasion and subsequent metastasis is the major cause of death from most cancers including prostate cancer. Herein we report on the potential tumor suppressive properties of Rab7, a GTPase that regulates trafficking of lysosomes. The movement of lysosomes to the cell surface in response to environmental cues increases the secretion of proteinases and cell invasion. We determined that Troglitazone and other members of the Thiazolidinedione family inhibit cell-surface directed lysosome trafficking and cathepsin B secretion through a Rab7-dependent mechanism. Moreover, Rab7 shRNA expressing cells were found to be more invasive in vitro and in vivo. Increased invasiveness was accompanied by elevated expression of the c-Met receptor and prolonged downstream signaling, thereby supporting a role for Rab7 as a mediator of signaling down-regulation. Taken together, these results suggested that Rab7 acts as a negative regulator of prostate tumor growth and invasion, providing further evidence for its potential as a tumor suppressor.

Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

Rapamycin inhibits lymphatic endothelial cell tube formation by downregulating vascular endothelial growth factor receptor 3 protein expression.

Mammalian target of rapamycin (mTOR) controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1)- or fetal bovine serum (FBS)-stimulated lymphatic endothelial cell (LEC) tube formation, an in vitro model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3) by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.

Guided visualization interventions on perceived stress, dyadic satisfaction and psychological symptoms in highly stressed couples.

This study focused on the effect of a brief CBT-based relaxation/guided visualization intervention on perceived stress, dyadic satisfaction and psychological symptoms. This study also tested the hypothesis that indicators of global orientation including Sense of Coherence and Differentiation of Self would mediate between perceived stress and symptoms of physical, psychological and relational distress. A three session intervention used guided visualization scripts which incorporated relaxation and controlled breathing techniques as well as a CBT approach that focused on stress management and internal locus of control. Results indicated that after the intervention, participants showed lower levels of perceived stress and lower levels of dyadic distress, as well as lower psychological and physical complaints as reported with the Symptom Checklist-90-R. Both Sense of Coherence and Differentiation of Self were mediators for the effect of perceived stress on the number of endorsed symptoms.

Association of core-binding factor ß with the malignant phenotype of prostate and ovarian cancer cells.

Core binding factor (CBF) is a transcription factor complex that plays roles in development, stem-cell homeostasis, and human disease. CBF is a heterodimer composed of one of three DNA-binding RUNX proteins plus the non-DNA-binding protein, CBFß. Recent studies have showed that the RUNX factors exhibit complex expression patterns in prostate, breast, and ovarian cancers, and CBF has been implicated in the control of cancer-related genes. However, the biologic roles of CBF in solid tumors have not been fully elucidated. To test whether CBF is required for the malignant phenotype of various epithelial cancers, we used lentiviral delivery of CBFß-specific shRNA to significantly decrease CBFß expression in two prostate cancer cell lines (PPC1 and PC-3) and the SKOV-3 ovarian cancer cell line. We found that knockdown of CBFß significantly inhibited anchorage independent growth of each cell line. Further, CBFß knockdown in PPC1 cells suppressed xenograft tumor growth compared to controls. Mice injected with SKOV-3 ovarian cancer cells knocked-down for CBFß exhibited a survival time similar to control mice. However, human cells recovered from the ascites fluid of these mice showed CBFß expression levels similar to those from mice injected with control SKOV-3 cells, suggesting that CBFß knockdown is incompatible with tumor cell growth. Gene expression profiling of CBFß knockdown cells revealed significant changes in expression in genes involved in various developmental and cell signaling pathways. These data collectively suggest that CBFß is required for malignancy in some human cancers.

Escherichia coli serotype O157:H7 retention on solid surfaces and peroxide resistance is enhanced by dual-strain biofilm formation.

In a previous study we showed that an Escherichia coli O157:H7 strain that was unable to form biofilm was retained in large numbers in dual-strain biofilms formed with an E. coli O-:H4 companion strain. In this study we tested additional companion strains for their ability to retain E. coli O157:H7 strain 0475s. Companion strains producing biofilm that withstood aggressive washes were able to significantly increase serotype O157:H7 retention. Dual-strain biofilms with certain companion strains retained higher percentages of strain 0475s, and that ability was independent of biofilm total cell numbers. Tests with additional non-biofilm-forming E. coli O157:H7 strains showed that enhancement by companion strains was not unique to strain 0475s. Experiments using an E. coli companion strain with deletions of various curli and cellulose genes indicated that dual-strain biofilm formation was dependent on companion strain properties. Strain 0475s was not able to generate biofilm or persist on plastic when grown in broth with a biofilm-forming companion and separated by a 0.2 microm porous membrane, indicating a requirement for intimate contact with the companion strain. When dual-strain biofilms and planktonic cells were challenged with 5% H(2)O(2), strain 0475 showed greater survival in biofilms with certain companion strains compared to the corresponding planktonic cells. The results of this study indicate that non-biofilm-forming E. coli O157:H7 strains are retained on solid surfaces associated with biofilms generated by companion strains. However, properties other than biofilm mass enable certain companion strains to retain greater numbers of E. coli O157:H7.

Design, synthesis and evaluation of novel 2-thiophen-5-yl-3H-quinazolin-4-one analogues as inhibitors of transcription factors NF-kappaB and AP-1 mediated transcriptional activation: Their possible utilization as anti-inflammatory and anti-cancer agents.

In an attempt to discover novel inhibitors of NF-kappaB and AP-1 mediated transcriptional activation utilizing the concept of chemical lead based medicinal chemistry and bioisosterism a series of 2-(2,3-disubstituted-thiophen-5-yl)-3H-quinazolin-4-one analogs was designed. A facile and simple route for the synthesis of the designed molecules was developed. Synthesized molecules were evaluated for their activity as inhibitors towards NF-kappaB and AP-1 mediated transcriptional activation in a cell line report-based assay. This series provides us with a substantial number of compounds inhibiting the activity of NF-kappaB and/or AP-1 mediated transcriptional activation. These compounds also exhibit anti-inflammatory and anti-cancer activity in in vivo models of inflammation and cancer. The 4-pyridyl group is found to be the most important pharmacophore on the third position of thiophene ring for inhibiting NF-kappaB and AP-1 mediated transcriptional activation. The relationships between the activities shown by these compounds in the in vivo and in vitro models have been established by using FVB transgenic mice model. These results suggest the suitability of the designed molecular framework as a potential scaffold for the design of molecules with inhibitory activity towards NF-kappaB and AP-1 mediated transcriptional activation, which may also exhibit anti-inflammatory and anti-cancer activity. This series of molecules warrants further study to explore their potential as therapies for use in chronic inflammatory conditions and cancer. Development of the synthetic protocol for the synthesis of this series of molecules, biological activities and a structure-activity relationship (SAR) have been discussed herein.

Design, synthesis and characterization of novel 2-(2,4-disubstituted-thiazole-5-yl)-3-aryl-3H-quinazoline-4-one derivatives as inhibitors of NF-kappaB and AP-1 mediated transcription activation and as potential anti-inflammatory agents.

A series of 2-(2,4-disubstituted-thiazole-5-yl)-3-aryl-3H-quinazoline-4-one derivatives were designed and synthesized. Synthesized molecules were further evaluated for their inhibitory activity towards transcription factors NF-kappaB and AP-1 mediated transcriptional activation in a cell line based in vitro assay as well as for their anti-inflammatory activity in in vivo model of acute inflammation. This series provides us with selective and dual inhibitors of NF-kappaB and AP-1 mediated transcriptional activation which also exhibit significant efficacy in in vivo model of inflammation. Two of the compounds 9m and 9o turned out to be the most promising dual inhibitors of NF-kappaB and AP-1 mediated transcriptional activation with an IC(50) of 3.3 microM for both. 9n (IC(50)=5.5 microM) and 9p (IC(50)=5.5 microM) emerged as selective inhibitors of NF-kappaB mediated transcriptional activation and 9c (IC(50)=5.5 microM) and 9d (IC(50)=5.5 microM) were found to be more selective inhibitor of AP-1 mediated transcriptional activity. Though the relationship between the activities shown by these compounds in in vivo and in vitro model is still to be established, these results suggest the suitability of the designed molecular framework as a potential anti-inflammatory molecular framework which also exhibits the inhibitory activity towards NF-kappaB and AP-1 mediated transcriptional activation. This will be worth studying further to explore its complete potential particularly in chronic inflammatory conditions. The structure activity relationship (SAR) of this series has been discussed herein.

High-efficiency particulate air filter test stand and aerosol generator for particle loading studies.

This manuscript describes the design, characterization, and operational range of a test stand and high-output aerosol generator developed to evaluate the performance of 30 x 30 x 29 cm(3) nuclear grade high-efficiency particulate air (HEPA) filters under variable, highly controlled conditions. The test stand system is operable at volumetric flow rates ranging from 1.5 to 12 standard m(3)/min. Relative humidity levels are controllable from 5%-90% and the temperature of the aerosol stream is variable from ambient to 150 degrees C. Test aerosols are produced through spray drying source material solutions that are introduced into a heated stainless steel evaporation chamber through an air-atomizing nozzle. Regulation of the particle size distribution of the aerosol challenge is achieved by varying source solution concentrations and through the use of a postgeneration cyclone. The aerosol generation system is unique in that it facilitates the testing of standard HEPA filters at and beyond rated media velocities by consistently providing, into a nominal flow of 7 standard m(3)/min, high mass concentrations (approximately 25 mg/m(3)) of dry aerosol streams having count mean diameters centered near the most penetrating particle size for HEPA filters (120-160 nm). Aerosol streams that have been generated and characterized include those derived from various concentrations of KCl, NaCl, and sucrose solutions. Additionally, a water insoluble aerosol stream in which the solid component is predominantly iron (III) has been produced. Multiple ports are available on the test stand for making simultaneous aerosol measurements upstream and downstream of the test filter. Types of filter performance related studies that can be performed using this test stand system include filter lifetime studies, filtering efficiency testing, media velocity testing, evaluations under high mass loading and high humidity conditions, and determination of the downstream particle size distributions.

Evaluation of a multi-component approach to cognitive-behavioral therapy (CBT) using guided visualizations, cranial electrotherapy stimulation, and vibroacoustic sound.

This pilot study examines the use of guided visualizations that incorporate both cognitive and behavioral techniques with vibroacoustic therapy and cranial electrotherapy stimulation to form a multi-component therapeutic approach. This multi-component approach to cognitive-behavioral therapy (CBT) was used to treat patients presenting with a range of symptoms including anxiety, depression, and relationship difficulties. Clients completed a pre- and post-session symptom severity scale and CBT skills practice survey. The program consisted of 16 guided visualizations incorporating CBT techniques that were accompanied by vibroacoustic therapy and cranial electrotherapy stimulation. Significant reduction in symptom severity was observed in pre- and post-session scores for anxiety symptoms, relationship difficulties, and depressive symptoms. The majority of the clients (88%) reported use of CBT techniques learned in the guided visualizations at least once per week outside of the sessions.

Bovine alveolar macrophage neurokinin-1 and response to substance P.

In this study bovine alveolar macrophage neurokinin-1 (NK-1) and the in vitro response to substance P (SP) exposure were investigated. Bovine alveolar macrophage membrane extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using anti-NK-1 antiserum demonstrated the presence of an approximately 60kDa band. Phagocytosis of fluorescent bioparticles by SP-exposed macrophages was 39% greater than that of non-exposed macrophages (P=0.0089). Likewise, there was 28% greater TNF production by macrophages following SP exposure compared to non-exposed controls (P=0.116). These results suggest that bovine alveolar macrophages respond to SP at least in part by enhancing phagocytosis and TNF production.