Exploring Pleiotropic Functions of Canine ß-Defensin 103: Nasal Cavity Expression, Antimicrobial Activity, and Melanocortin Receptor Activity.

Canine ß-defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a ß-defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient-rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient-rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus. As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin-4 receptor, acting as a weak allosteric agonist.

The Combination of Omega-3 Stearidonic Acid and Docetaxel Enhances Cell Death over Docetaxel Alone in Human Prostate Cancer Cells.

Background: Docetaxel (DOC), or Taxotere, is an anthracycline antibiotic used to treat multiple types of cancer. It is a first-line chemotherapy treatment for patients with metastasized, hormone-resistant prostate cancer (PCa) or for patients with high-risk, localized PCa that could benefit from early chemotherapy treatment. Previously, we showed that stearidonic acid (SDA), an omega-3 fatty acid, enhances the cytotoxicity of doxorubicin (DOX) in human PCa cells. This observation suggests that PCa therapies using SDA and chemotherapeutic drugs in combination offer attractive possibilities for developing treatments that ameliorate toxic side effects of some commonly used chemotherapy drugs. Objectives: We used androgen-resistant PC3 and DU 145 cells derived from human prostate cancer to quantify the effects of combined SDA and DOC on proliferation/viability and on the production of pro-apoptotic caspases 9 and 3. We also compared the effects of SDA with those of BAY, a pharmacological inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), in androgen-sensitive LNCaP cells. Finally, we qualitatively and quantitatively assessed the drug combination on androgen receptor (AR) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in LNCaP and PC3 cells, respectively. Methods: The half maximal inhibitory concentration (IC50) and combination indices of SDA and DOC in PC3 and DU 145 cells were determined using the MTT cell viability assay. To quantify the effects of SDA and BAY on NF-ĸB activity, we used luciferase reporter assays in LNCaP cells that were stably transduced with lentiviral vectors carrying NF-ĸB response element sequence upstream of the luciferase gene sequence. AR and PPARγ expression were assessed by western blotting and immunocytochemistry. We considered caspase 9 and 3 cleavage to be apoptosis markers and determined the drug combination effect on the extent of that cleavage by western blot analysis. Results: The cytotoxic effects of DOC were synergistically enhanced by SDA when the two were added to DU145 and PC3 cell cultures. Combination index (CI) analyses based on the Chou-Talalay method and mass action law showed synergistic interaction with CI <1. SDA suppressed TNFα-induced NF-κB activity similarly to BAY. The SDA/DOC combination down regulated testosterone (T)-induced AR and troglitazone-induced PPARγ protein expression when compared to using the drugs singly. Similarly, the SDA/DOC combination induced caspase 9 and 3 production and cleavage suggesting apoptosis induction. Like our DOX studies, this work provides proof-of-concept for using SDA and DOC in combination to reduce the dose, and therefore the toxicity, of DOC and possibly increasing the survival benefit in DOC clinical translation studies.

AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease.

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme ß-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

Expression of melanocortin receptors in human prostate cancer cell lines: MC2R activation by ACTH increases prostate cancer cell proliferation.

The melanocortin receptors (MCRs 1-5) are G protein coupled-receptors (GPCRs) that regulate food intake, inflammation, skin pigmentation, sexual function and steroidogenesis. Their peptide ligands, the melanocortins, are α-, ß- and γ-melanocyte-stimulating hormone and adrenocorticotropic hormone (ACTH) all of which are secreted from the anterior pituitary gland under hypothalamic control. MC2R binds ACTH but has no affinity for the other melanocortins and is, thereby, pharmacologically different from MCRs that bind those ligands. Evidence suggests that elevated GPCRs transactivate the androgen receptor (AR), the critical mediator of prostate cell growth, and consequently promote prostate cancer cell proliferation. It may be that reduced central melanocortin signaling is coincidental with reversal of prostate cancer cachexia, but no data are available on the expression of, or the role for, MCRs in prostate cancer. Here, we show that MCR (1-5) mRNAs are expressed in androgen-dependent LNCaP and androgen-independent PC3 and DU-145 human prostate cancer cell lines. Further, MC2R, the specific target of ACTH, is expressed in LNCaP, PC3 and DU-145 cells. Among the several synthetic MCR peptide ligands that we used, only ACTH promoted concentration-dependent cell proliferation in the three cell lines as shown by MTT cell proliferation assay. In LNCaP cells, the effect was additive with testosterone stimulation and was partially blunted with SHU9119, a non-selective MCR antagonist. In the same cells, ACTH induced cAMP production and increased AR nuclear labeling in immunocytochemical assays. Our observations suggest that MC2R is involved in prostate carcinogenesis and that targeting MC2R signaling may provide a novel avenue in prostate carcinoma treatment.

Olfactory responses to explosives associated odorants are enhanced by zinc nanoparticles.

Many odorants related to manufactured explosives have low volatilities and are barely detectable as odors. We previously reported that zinc metal nanoparticles increased rat olfactory epithelium responses, measured by electroolfactogram (EOG), to several odorants. Here, we report that nanomolar concentrations of zinc metal nanoparticles strongly enhanced olfactory responses to the explosives related odorants cyclohexanone, methyl benzoate, acetophenone, and eugenol. Rat olfactory epithelium was exposed to metal nanoparticles and odorant responses were quantified by EOG. Zinc nanoparticles added to explosive odorants strongly increased the odorant response in a dose-dependent manner. The enzymatic breakdown of the second messenger cyclic adenosine monophosphate (cAMP) was prevented by adding the membrane-permeable phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This caused the olfactory cilia cAMP concentration to increase and generated EOG signals. The EOG responses generated by IBMX were not enhanced by zinc nanoparticles. Based on these observations, we conclude that zinc nanoparticles act at the receptor site and are involved in the initial events of olfaction. Our results suggest that zinc metal nanoparticles can be used to facilitate a canine detection of explosive odorants.

Olfactory marker protein expression in the vomeronasal neuroepithelium of tamarins (Saguinus spp).

Knowledge of the vomeronasal neuroepithelium (VNNE) microanatomy is disproportionately based on rodents. To broaden our knowledge, we examined olfactory marker protein (OMP) expression in a sample of twenty-three non-human primates. The density of OMP (+) vomeronasal sensory neurons (VSNs) in the VNNE was measured. Here we compared OMP (+) VSN density in five species of Saguinus (a genus of New World monkey) of different ages to a comparative primate sample that included representatives of every superfamily in which a VNO is postnatally present. In Saguinus spp., the VNNE at birth is thin, usually comprising one or two nuclear rows. At all ages studied, few VNNE cells are OMP reactive as view in coronal sections. In the comparative sample, the OMP (+) VSNs appear to be far more numerous in the spider monkey (another New World monkey) and the bushbaby (a distant relative). Other species (e.g., owl monkey) had a similar low density of OMP (+) VSNs as in Saguinus. These results expand our earlier finding that few VSNs are OMP (+) in Saguinus geoffroyi to other species of the genus. Our sample indicates that the number of OMP (+) VSNs in primates varies from ubiquitous to few with New World monkeys varying the most. The scarcity of OMP (+) cells in some primate VNOs reflects a lower number of terminally differentiated VSNs compared to a diverse range of mammals. If primates with relatively few OMP (+) VSNs have a functional vomeronasal system, OMP is not critical for stimulus detection.

Bovine viral diarrhea virus is a suitable viral vector for stable expression of heterologous gene when inserted in between N(pro) and C genes.

Bovine viral diarrhea virus (BVDV) is a group of small enveloped viruses with a single-stranded, positive-oriented RNA genome of approximately 12.3 kb. BVDV genome directs the production of a viral polyprotein that is subsequently cleaved to release the mature viral proteins. To explore the potential of using BVDV as viral vector for stable expression of heterologous genes, eGFP2A was inserted in between N(pro) and C genes of a noncytopathic type-I BVDV strain SD1. eGFP2A was designed with eGFP protein in frame fused to the N terminus of the foot-and-mouth disease virus 2A protease. This strategy promised not only the correct processing of both viral N(pro) and C protein but also releasing of the chimeric protein from the nascent viral polyprotein. The recombinant reporter virus was successfully rescued in MDBK cells. In vitro study showed that eGFP2A protein, as expected, was expressed and processed properly from the nascent viral polyprotein. The reporter virus was similar to wt SD1 in viral RNA replication and protein expression and comparable to wt SD1 in growth kinetics except that this virus had a peak virus titer approximately 0.5 log(10) lower and a maximum yield about 4h later than wt SD1. In summary, these results indicated that BVDV is a suitable viral vector for stable expression of heterologous genes when inserted in between N(pro) and C genes.

Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor gamma with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development.

Exposure to the estrogen receptor alpha (ERalpha) ligand diethylstilbesterol (DES) between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ERalpha and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Transcripts for PPARs alpha, beta, and gamma and gamma1a splice variant were detected in Sprague-Dawley normal rat penis with PPARgamma predominating. In addition, PPARgamma1b and PPARgamma2 were newly induced by DES. The PPARgamma transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPARgamma protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ERalpha and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPARgamma expression. These results suggest a biological overlap between PPARgamma and ERalpha and highlight a mechanism for endocrine disruption.

Temporal delay of peak T-cell immunity determines Chlamydia pneumoniae pulmonary disease in mice.

Severe chlamydial disease typically occurs after previous infections and results from a hypersensitivity response that is also required for chlamydial elimination. Here, we quantitatively dissected the immune and disease responses to repeated Chlamydia pneumoniae lung infection by multivariate modeling with four dichotomous effects: mouse strain (A/J or C57BL/6), dietary protein content (14% protein and 0.3% L-cysteine-0.9% L-arginine, or 24% protein and 0.5% L-cysteine-2.0% L-arginine), dietary antioxidant content (90 IU alpha-tocopherol/kg body weight versus 450 IU alpha-tocopherol/kg and 0.1% g L-ascorbate), and time course (3 or 10 days postinfection). Following intranasal C. pneumoniae challenge, C57BL/6 mice on a low-protein/low-antioxidant diet, but not C57BL/6 mice on other diets or A/J mice, exhibited profoundly suppressed early lung inflammatory and pan-T-cell (CD3delta(+)) and helper T-cell (CD45) responses on day 3 but later strongly exacerbated disease on day 10. Contrast analyses characterized severe C. pneumoniae disease as being a delayed-type hypersensitivity (DTH) response with increased lung macrophage and Th1 cell marker transcripts, increased Th1:Th2 ratios, and Th1 cytokine-driven inflammation. Results from functional analyses by DTH, enzyme-linked immunospot, and immunohistofluorescence assays were consistent with the results obtained by transcript analysis. Thus, chlamydial disease after secondary infection is a temporal dysregulation of the T-cell response characterized by a profoundly delayed T-helper cell response that results in a failure to eliminate the pathogen and provokes later pathological Th1 inflammation. This delayed T-cell response is under host genetic control and nutritional influence. The mechanism that temporally and quantitatively regulates the host T-cell population is the critical determinant in chlamydial pathogenesis.