Glycemic Challenge Is Associated with the Rapid Cellular Activation of the Locus Ceruleus and Nucleus of Solitary Tract: Circumscribed Spatial Analysis of Phosphorylated MAP Kinase Immunoreactivity.

Rodent studies indicate that impaired glucose utilization or hypoglycemia is associated with the cellular activation of neurons in the medulla (Winslow, 1733) (MY), believed to control feeding behavior and glucose counterregulation. However, such activation has been tracked primarily within hours of the challenge, rather than sooner, and has been poorly mapped within standardized brain atlases. Here, we report that, within 15 min of receiving 2-deoxy-d-glucose (2-DG; 250 mg/kg, i.v.), which can trigger glucoprivic feeding behavior, marked elevations were observed in the numbers of rhombic brain (His, 1893) (RB) neuronal cell profiles immunoreactive for the cellular activation marker(s), phosphorylated p44/42 MAP kinases (phospho-ERK1/2), and that some of these profiles were also catecholaminergic. We mapped their distributions within an open-access rat brain atlas and found that 2-DG-treated rats (compared to their saline-treated controls) displayed greater numbers of phospho-ERK1/2+ neurons in the locus ceruleus (Wenzel and Wenzel, 1812) (LC) and the nucleus of solitary tract (>1840) (NTS). Thus, the 2-DG-activation of certain RB neurons is more rapid than perhaps previously realized, engaging neurons that serve multiple functional systems and which are of varying cellular phenotypes. Mapping these populations within standardized brain atlas maps streamlines their targeting and/or comparable mapping in preclinical rodent models of disease.

Does acute heat stress differentially-modulate expression of ionotropic neurotransmitter receptors in the RVLM of young and aged F344 rats?

The rostral ventral lateral medulla (RVLM) is a brainstem area that plays a role in regulating numerous physiological systems, especially their responsiveness to acute stress. Aging affects the responsiveness of RVLM neural circuits to acute stress. Based on the relationship between ionotropic neurotransmitter receptors in the RVLM and the physiological functions mediated via activation of these receptors, we hypothesized that in response to acute heat stress the expression of ionotropic neurotransmitter receptors in the RVLM of aged rats would be characterized by upregulation of inhibitory subunits and downregulation of excitatory subunits. The goal of the present study was to determine the effect of acute heating on the gene expression profile of RVLM inhibitory (GABAA and Glycine) and excitatory (NMDA and AMPA) ionotropic neurotransmitter receptor subunits in young and aged F344 rats. RVLM tissue punches from young and aged F344 rats were analyzed using TaqMan qPCR and immunoblotting. When compared to age-matched controls, heat stress increased the gene expression of RVLM inhibitory receptor subunits in aged (Gabra1, Gabra2, Gabra5, Glra1) and young (Gabra1) F344 rats at mRNA level, with little change in the expression of RVLM excitatory receptor subunits. Significant age x heat interaction effects were observed with increased expression of Gabra2 and Gabrb1 inhibitory receptor subunits and decreased expression of Gria1 and Gria2 excitatory receptor subunits in the RVLM of aged F344 rats, with the most marked change observed with the Gabra2 subunit, which was validated by immunoblotting. These findings demonstrate that in response to acute heat stress there is enhanced expression of inhibitory ionotropic receptor subunits in aged compared to young rats, supporting the idea that advanced age may alter RVLM responsivity by affecting the molecular substrate of ionotropic receptors.

Microarray analysis of aging-associated immune system alterations in the rostral ventrolateral medulla of F344 rats.

The rostral ventrolateral medulla (RVLM) is an area of the brain stem that contains diverse neural substrates that are involved in systems critical for physiological function. There is evidence that aging affects some neural substrates within the RVLM, although age-related changes in RVLM molecular mechanisms are not well established. The goal of the present study was to characterize the transcriptomic profile of the aging RVLM and to test the hypothesis that aging is associated with altered gene expression in the RVLM, with an emphasis on immune system associated gene transcripts. RVLM tissue punches from young, middle-aged, and aged F344 rats were analyzed with Agilent's whole rat genome microarray. The RVLM gene expression profile varied with age, and an association between chronological age and specific RVLM gene expression patterns was observed [P < 0.05, false discovery rate (FDR) < 0.3]. Functional analysis of RVLM microarray data via gene ontology profiling and pathway analysis identified upregulation of genes associated with immune- and stress-related responses and downregulation of genes associated with lipid biosynthesis and neurotransmission in aged compared with middle-aged and young rats. Differentially expressed genes associated with the complement system and microglial cells were further validated by quantitative PCR with separate RVLM samples (P < 0.05, FDR < 0.1). The present results have identified age-related changes in the transcriptomic profile of the RVLM, modifications that may provide the molecular backdrop for understanding age-dependent changes in physiological regulation.

Does aging alter the molecular substrate of ionotropic neurotransmitter receptors in the rostral ventral lateral medulla? - A short communication.

Aging alters sympathetic nervous system (SNS) regulation, although central mechanisms are not well understood. In young rats the rostral ventral lateral medulla (RVLM) is critically involved in central SNS regulation and RVLM neuronal activity is mediated by a balance of excitatory and inhibitory ionotropic neurotransmitters and receptors, providing the foundation for hypothesizing that with advanced age the molecular substrate of RVLM ionotropic receptors is characterized by upregulated excitatory and downregulated inhibitory receptor subunits. This hypothesis was tested by comparing the relative mRNA expression and protein concentration of RVLM excitatory (NMDA and AMPA) and inhibitory (GABA and glycinergic) ionotropic neurotransmitter receptor subunits in young and aged Fischer (F344) rats. Brains were removed from anesthetized rats and the RVLM-containing area was micropunched and extracted RNA and protein were subsequently used for TaqMan qRT-PCR gene expression and quantitative ELISA analyses. Bilateral chemical inactivation of RVLM neurons and peripheral ganglionic blockade on visceral sympathetic nerve discharge (SND) was determined in additional experiments. The relative gene expression of RVLM NMDA and AMPA glutamate-gated receptor subunits and protein concentration of select receptor subunits did not differ between young and aged rats, and there were no age-related differences in the expression of RVLM ionotropic GABAA and Gly receptors, or of protein concentration of select GABAA subunits. RVLM muscimol microinjections significantly reduced visceral SND by 70±2% in aged F344 rats. Collectively these findings from this short communication support a functional role for the RVLM in regulation of sympathetic nerve outflow in aged rats, but provide no evidence for an ionotropic RVLM receptor-centric framework explaining age-associated changes in SNS regulation.

Effect of ghrelin on regulation of splenic sympathetic nerve discharge.

Ghrelin influences immune system function and modulates the sympathetic nervous system; however, the contribution of ghrelin to neural-immune interactions is not well-established because the effect of ghrelin on splenic sympathetic nerve discharge (SND) is not known. This study tested the hypothesis that central ghrelin administration would inhibit splenic SND in anesthetized rats. Rats received intracerebroventricular (ICV) injections of ghrelin (1nmol/kg) or aCSF. Lumbar SND recordings provided a non-visceral nerve control. The ICV ghrelin administration significantly increased splenic and lumbar SND, whereas mean arterial pressure (MAP) was not altered. These findings provide fundamental information regarding the nature of sympathetic-immune interactions.

A nanosensor for ultrasensitive detection of oversulfated chondroitin sulfate contaminant in heparin.

Heparin has been extensively used as an anticoagulant for the last eight decades. Recently, the administration of a contaminated batch of heparin caused 149 deaths in several countries including USA, Germany, and Japan. The contaminant responsible for the adverse effects was identified as oversulfated chondroitin sulfate (OSCS). Here, we report a rapid, ultrasensitive method of detecting OSCS in heparin using a nanometal surface energy transfer (NSET) based gold-heparin-dye nanosensor. The sensor is an excellent substrate for heparitinase enzyme, as evidenced by ~70% recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, as the enzyme is inhibited by the contaminant. The newly designed nanosensor can detect as low as 1 × 10(-9) % (w/w) OSCS making it the most sensitive tool to date for the detection of trace amounts of OSCS in pharmaceutical heparins.

Luminol-based bioluminescence imaging of mouse mammary tumors.

Polymorphonuclear neutrophils (PMNs) are the most abundant circulating blood leukocytes. They are part of the innate immune system and provide a first line of defense by migrating toward areas of inflammation in response to chemical signals released from the site. Some solid tumors, such as breast cancer, also cause recruitment and activation of PMNs and release of myeloperoxidase. In this study, we demonstrate that administration of luminol to mice that have been transplanted with 4T1 mammary tumor cells permits the detection of myeloperoxidase activity, and consequently, the location of the tumor. Luminol allowed detection of activated PMNs only two days after cancer cell transplantation, even though tumors were not yet palpable. In conclusion, luminol-bioluminescence imaging (BLI) can provide a pathway towards detection of solid tumors at an early stage in preclinical tumor models.

Luminol-labeled gold nanoparticles for ultrasensitive chemiluminescence-based chemical analyses.

We report a study on chemiluminescence-based chemical analyses using luminol molecules covalently attached to 10 nm diameter gold nanoparticles (GNPs). Chemiluminescence (CL) has been systematically studied under two schemes by varying the concentrations of luminol-labeled GNPs and [Fe(CN)6](3-) catalyst, respectively. The CL signal of luminol-labeled GNPs is enhanced by 5 to 10 times compared to the bulk luminol solutions of the same concentration. The log-log plot of the CL signal versus the number of luminol-labeled GNPs suspended in a standard 96-well plate shows two characteristic linear curves with distinct slopes across eight orders of magnitude variation in the GNP quantity (from 1.82 × 10(2) to 1.82 × 10(10) GNPs per well). The detection limit represented by the cross-point of these two curves can reach down to ~6.1 × 10(5) GNPs per well (corresponding to 1.0 × 10(-14) M GNP and 2.4 × 10(-11) M equivalent luminol concentration). The attachment of luminol molecules to GNP nano-carriers allows a large amount of luminol to be placed in a greatly reduced volume (or area) toward developing miniaturized CL sensors. We have demonstrated this by preloading dried luminol-labeled GNPs in homemade microwell arrays (with a volume of ~12 µL per well). A linear log-log curve can be obtained across the full range from 1 × 10(3) to 1 × 10(10) GNPs per microwell. The CL signal was detectable with as few as ~1000 GNPs. We have further applied this microwell method to the detection of highly diluted blood samples, in both intact and lysed forms, which releases Fe(3+)-containing hemoglobin to catalyze luminol CL. The lysed blood sample can be detected even after a 10(8) fold dilution (corresponding to ~0.18 cells per well). This ultrasensitive CL detection method may be readily adapted for developing various miniaturized multiplex biosensors for rapid chemical/biochemical analyses.

Magnetic-Fe/Fe(3)O(4)-nanoparticle-bound SN38 as carboxylesterase-cleavable prodrug for the delivery to tumors within monocytes/macrophages.

The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe(3)O(4) magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system.

Stem cell-based photodynamic therapy.

We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.

Interleukin-1ß and transforming growth factor-ß cooperate to induce neurosphere formation and increase tumorigenicity of adherent LN-229 glioma cells.

INTRODUCTION: Glioma stem cells (GSCs) have the property of self-renewal and appear to be a driving force for the initiation and recurrence of gliomas. We recently found that the human tumorigenic LN-229 glioma cell line failed to form neurospheres in serum-free conditions and generated mostly small tumors in vivo, suggesting that either LN-229 GSCs are not active in these conditions or GSCs are absent in the LN-229 cell line. METHODS: Using self-renewal assay, soft-agar colony assay, cell proliferation assay, invasion assay, real time PCR analysis, ELISA and in vivo tumorigenic assay, we investigated the effects of interleukin (IL)-1ß and transforming growth factor (TGF)-ß on the development of GSCs from LN-229 cells. RESULTS: Here, we demonstrate that the combination of IL-1ß and TGF-ß can induce LN-229 cells to form neurospheres in serum-free medium. IL-1ß/TGF-ß-induced neurospheres display up-regulated expression of stemness factor genes (nestin, Bmi-1, Notch-2 and LIF), and increased invasiveness, drug resistance and tumor growth in vivo: hallmarks of GSCs. These results indicate that IL-1ß and TGF-ß cooperate to induce a GSC phenotype in the LN-229 cell line. Induction of nestin, LIF and Notch-2 by IL-1ß/TGF-ß can be reverted after cytokine withdrawal. Remarkably, however, up-regulated Bmi-1 levels remained unchanged after cytokine withdrawal; and the cytokine-withdrawn cells maintained strong clonogenicity, suggesting that Bmi-1 may play a crucial role in tumorigenesis. CONCLUSIONS: Our finding indicates that glioma cells without self-renewal capability in standard conditions could also contribute to glioma malignancy when cytokines, such as IL-1ß and TGF-ß, are present in the tumor environment. Targeting GSC-promoting cytokines that are highly expressed in glioblastomas may contribute to the development of more effective glioma therapies.

A cell-delivered and cell-activated SN38-dextran prodrug increases survival in a murine disseminated pancreatic cancer model.

Enzyme-activated prodrugs have been investigated and sought after as highly specific, low-side-effect treatments, especially for cancer therapy. Unfortunately, excellent targets for enzyme-activated therapy are rare. Here a system based on cell delivery that can carry both a prodrug and an activating enzyme to the cancer site is demonstrated. Raw264.7 cells (mouse monocyte/macrophage-like cells, Mo/Ma) are engineered to express intracellular rabbit carboxylesterase (InCE), which is a potent activator of the prodrug irinotecan to SN38. InCE expression is regulated by the TetOn® system, which silences the gene unless a tetracycline, such as doxycycline, is present. Concurrently, an irinotecan-like prodrug, which is conjugated to dextran and can be loaded into the cytoplasm of Mo/Ma, is synthesized. To test the system, a murine pancreatic cancer model is generated by intraperitoneal (i.p.) injection of Pan02 cells. Engineered Mo/Ma are loaded with the prodrug and are injected i.p. Two days later, doxycycline was given i.p. to activate InCE, which activated the prodrug. A survival study demonstrates that this system significantly increased survival in a murine pancreatic cancer model. Thus, for the first time, a prodrug/activating enzyme system, which is self-contained within tumor-homing cells and can prolong the life of i.p. pancreatic tumor bearing mice, is demonstrated.

Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer model.

Using magnetic nanoparticles to absorb alternating magnetic field energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment. This report demonstrates a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for alternating magnetic field treatment. Paramagnetic iron/ iron oxide nanoparticles were synthesized and loaded into RAW264.7 cells (mouse monocyte/ macrophage-like cells), which have been shown to be tumor homing cells. A murine model of disseminated peritoneal pancreatic cancer was then generated by intraperitoneal injection of Pan02 cells. After tumor development, monocyte/macrophage-like cells loaded with iron/ iron oxide nanoparticles were injected intraperitoneally and allowed to migrate into the tumor. Three days after injection, mice were exposed to an alternating magnetic field for 20 minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-tumor insertion life expectancy increase of 31%. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer.

A self-contained enzyme activating prodrug cytotherapy for preclinical melanoma.

Gene-directed enzyme prodrug therapy (GDEPT) has been investigated as a means of cancer treatment without affecting normal tissues. This system is based on the delivery of a suicide gene, a gene encoding an enzyme which is able to convert its substrate from non-toxic prodrug to cytotoxin. In this experiment, we have developed a targeted suicide gene therapeutic system that is completely contained within tumor-tropic cells and have tested this system for melanoma therapy in a preclinical model. First, we established double stable RAW264.7 monocyte/macrophage-like cells (Mo/Ma) containing a Tet-On® Advanced system for intracellular carboxylesterase (InCE) expression. Second, we loaded a prodrug into the delivery cells, double stable Mo/Ma. Third, we activated the enzyme system to convert the prodrug, irinotecan, to the cytotoxin, SN-38. Our double stable Mo/Ma homed to the lung melanomas after 1 day and successfully delivered the prodrug-activating enzyme/prodrug package to the tumors. We observed that our system significantly reduced tumor weights and numbers as targeted tumor therapy after activation of the InCE. Therefore, we propose that this system may be a useful targeted melanoma therapy system for pulmonary metastatic tumors with minimal side effects, particularly if it is combined with other treatments.

Human xenografts are not rejected in a naturally occurring immunodeficient porcine line: a human tumor model in pigs.

Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments; however, therapies tested in such models often fail to translate into clinical settings. Therefore, a better preclinical model for cancer treatment testing is needed. Here we demonstrate that an immunodeficient line of pigs can host and support the growth of xenografted human tumors and has the potential to be an effective animal model for cancer therapy. Wild-type and immunodeficient pigs were injected subcutaneously in the left ear with human melanoma cells (A375SM cells) and in the right ear with human pancreatic carcinoma cells (PANC-1). All immunodeficient pigs developed tumors that were verified by histology and immunohistochemistry. Nonaffected littermates did not develop tumors. Immunodeficient pigs, which do not reject xenografted human tumors, have the potential to become an extremely useful animal model for cancer therapy because of their similarity in size, anatomy, and physiology to humans.

Attenuation of mouse melanoma by A/C magnetic field after delivery of bi-magnetic nanoparticles by neural progenitor cells.

Localized magnetic hyperthermia as a treatment modality for cancer has generated renewed interest, particularly if it can be targeted to the tumor site. We examined whether tumor-tropic neural progenitor cells (NPCs) could be utilized as cell delivery vehicles for achieving preferential accumulation of core/shell iron/iron oxide magnetic nanoparticles (MNPs) within a mouse model of melanoma. We developed aminosiloxane-porphyrin functionalized MNPs, evaluated cell viability and loading efficiency, and transplanted neural progenitor cells loaded with this cargo into mice with melanoma. NPCs were efficiently loaded with core/shell Fe/Fe(3)O(4) MNPs with minimal cytotoxicity; the MNPs accumulated as aggregates in the cytosol. The NPCs loaded with MNPs could travel to subcutaneous melanomas, and after A/C (alternating current) magnetic field (AMF) exposure, the targeted delivery of MNPs by the cells resulted in a measurable regression of the tumors. The tumor attenuation was significant (p < 0.05) a short time (24 h) after the last of three AMF exposures.

A/C magnetic hyperthermia of melanoma mediated by iron(0)/iron oxide core/shell magnetic nanoparticles: a mouse study.

BACKGROUND: There is renewed interest in magnetic hyperthermia as a treatment modality for cancer, especially when it is combined with other more traditional therapeutic approaches, such as the co-delivery of anticancer drugs or photodynamic therapy. METHODS: The influence of bimagnetic nanoparticles (MNPs) combined with short external alternating magnetic field (AMF) exposure on the growth of subcutaneous mouse melanomas (B16-F10) was evaluated. Bimagnetic Fe/Fe3O4 core/shell nanoparticles were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected against rapid biocorrosion by organic dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin) units were attached to the dopamine-oligoethylene glycol ligands. RESULTS: The magnetic hyperthermia results obtained after intratumoral injection indicated that micromolar concentrations of iron given within the modified core-shell Fe/Fe3O4 nanoparticles caused a significant anti-tumor effect on murine B16-F10 melanoma with three short 10-minute AMF exposures. We also observed a decrease in tumor size after intravenous administration of the MNPs followed by three consecutive days of AMF exposure 24 hrs after the MNPs injection. CONCLUSIONS: These results indicate that intratumoral administration of surface modified MNPs can attenuate mouse melanoma after AMF exposure. Moreover, we have found that after intravenous administration of micromolar concentrations, these MNPs are capable of causing an anti-tumor effect in a mouse melanoma model after only a short AMF exposure time. This is a clear improvement to state of the art.