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Animal models are essential to cancer research, but current xenograft models are limited in their utility especially due to the lack of an immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and after birth mice were challenged with A375 cells to determine their ability to develop tumors. Intravenous injections of cells resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. These results were replicated with several other human tumor types: BxPC3 (human pancreatic adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma), M21 (human melanoma), and HeLa (human cervical adenocarcinoma). Development of an immunocompetent xenograft tumor model would allow the further elucidation of the interaction of the immune system with therapy in both preclinical research and patient derived xenografts.

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.

Objective: To investigate the feasibility and efficacy of localized, subtotal, cortical-sparing microwave thermal ablation (MTA) as a potential curative management for primary aldosteronism. The study investigated with equal importance the selected ablation of small volumes of adrenal cortex while sparing adjacent cortex. Method: An in-vivo study was carried out in swine (n = 8) where MTA was applied under direct visualization, to the adrenal glands at 45 W or 70 W for 60 s, using a lateral, side-firing probe and a non-penetrative approach. Animals were survived for 48 h post-procedurally. Animals were investigated for markers of histological, immunohistochemical and biochemical evidence of adrenal function and adrenal damage by assessing samples drawn intra-operatively and at the time of euthanasia. Results: Selected MTA (70 W for 60 s) successfully ablated small adrenocortical volumes (∼0.8 cm3) characterized by coagulative necrosis and abnormal expression of functional markers (CYP11B1 and CYP17). Non-ablated, adjacent cortex was not affected and preserved normal expression of functional markers, without increased expression of markers of heat damage (HSP-70 and HMGB-1). Limited adrenal medullary damage was demonstrated histologically, clinically and biochemically. Conclusion: MTA offers potential as an efficient methodology for delivering targeted subtotal cortical-sparing adrenal ablation. Image-guided targeted MTA may also represent a safe future modality for curative management of PA, in the setting of both unilateral and bilateral disease.

Esophageal varices are a significant complication of portal hypertension. Endoscopic variceal ligation (EVL) is one of the clinical standards for treating these varices and preventing their hemorrhage. Limitations of EVL include the risk of stricture formation and postband ulcer bleeding due to the damage caused to the esophageal mucosa, as well as the need for multiple endoscopic treatment sessions to eradicate the varices. The goal of this study is to develop a device and evaluate the technical feasibility of microwave ablation to seal esophageal varices, while preventing thermal damage to the surface mucosal tissue. A microwave applicator with a directional radiation pattern was developed for endoscopic ablation of esophageal varices. Electromagnetic and bioheat transfer computational models were employed to optimize the design of the microwave applicator and evaluate energy delivery strategies for this application. Experiments in ex vivo and in vivo tissue were employed to verify simulation results. Simulations predicted enhanced heating performance of the antenna using an angled monopole radiating element. Further, simulations indicate that while the endoscopic cap attenuated electric fields in tissue, it also enhanced surface cooling of tissue, increasing the likelihood of preserving mucosal tissue. Experiments in ex vivo tissue indicated the feasibility of sealing veins with 77 W microwave power delivered for 30 s. In vivo experiments demonstrated the ability to seal veins, while preserving surface tissue. This study demonstrated the technical feasibility of microwave thermal ablation for treating esophageal varices using a 2.45 GHz water-cooled directional microwave applicator.

The present review assesses the current state of literature defining integrative autonomic-immune physiological processing, focusing on studies that have employed electrophysiological, pharmacological, molecular biological, and central nervous system experimental approaches. Central autonomic neural networks are informed of peripheral immune status via numerous communicating pathways, including neural and non-neural. Cytokines and other immune factors affect the level of activity and responsivity of discharges in sympathetic and parasympathetic nerves innervating diverse targets. Multiple levels of the neuraxis contribute to cytokine-induced changes in efferent parasympathetic and sympathetic nerve outflows, leading to modulation of peripheral immune responses. The functionality of local sympathoimmune interactions depends on the microenvironment created by diverse signaling mechanisms involving integration between sympathetic nervous system neurotransmitters and neuromodulators; specific adrenergic receptors; and the presence or absence of immune cells, cytokines, and bacteria. Functional mechanisms contributing to the cholinergic anti-inflammatory pathway likely involve novel cholinergic-adrenergic interactions at peripheral sites, including autonomic ganglion and lymphoid targets. Immune cells express adrenergic and nicotinic receptors. Neurotransmitters released by sympathetic and parasympathetic nerve endings bind to their respective receptors located on the surface of immune cells and initiate immune-modulatory responses. Both sympathetic and parasympathetic arms of the autonomic nervous system are instrumental in orchestrating neuroimmune processes, although additional studies are required to understand dynamic and complex adrenergic-cholinergic interactions. Further understanding of regulatory mechanisms linking the sympathetic nervous, parasympathetic nervous, and immune systems is critical for understanding relationships between chronic disease development and immune-associated changes in autonomic nervous system function.

A class of substituted 1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyrans (tricyclic pyrones; TPs) was synthesized from a one-pot condensation reaction of 6-substituted 4-hydroxy-2-pyrones and cyclohexenecarboxaldehydes. The reaction involves a 6pi-electrocyclic ring closing process, and stereo- and regioselectivities were examined. C3-Pyridyl-containing TPs may represent a novel synthetic class of microtubule de-stabilizing anti-cancer drugs that inhibit macromolecule synthesis, tubulin polymerization, and the proliferation of a spectrum of wild-type and multi-drug resistant tumor cell lines in vitro. A linear skeleton with a N-containing aromatic ring attached at C3 of the top A-ring, a central pyran B-ring and a six-membered bottom C-ring with no alkylation at C7 are required for the antitumor activities of the lead compounds, a 3-pyridyl benzopyran (code name H10) and its 2-pyridyl regioisomer (code name H19). In addition to interacting with the colchicine-binding site to inhibit tubulin polymerization and increase the mitotic index, these TP analogs also block the cellular transport of nucleosides to inhibit DNA synthesis more effectively than other antimitotic agents. The anticancer potential of TPs in vivo is suggested by the fact that i.p. injections of H10 decrease the growth of solid tumors in mice inoculated with lung or ovarian carcinomas. A drug-delivery system involving nanogels was studied. We incorporated the anticancer compound, 6-hydroxymethyl-1,4-anthracenedione (code name AQ10) into PEG-PEI nanogel, and found that AQ10-encapsulated nanogel PEG-PEI is significantly more effective in altering the growth of Pan 02 (pancreatic cancer) cells compared to AQ10 or nanogel PEG-PEI alone. Since AQ10 is insoluble in water, PEG-PEI encapsulation represents a way to solubilize and deliver this as well as other poorly soluble compounds.

Polyethylene glycol-polyethylenimine (PEG-PEI) nanogels have been used to deliver nucleic acids and oligonucleotides into cells. First, we synthesized PEG-PEI nanogels with methylene proton ratios (CH2O:CH2N) in PEG-PEI ranging from ∼6.8:1 to 4:1 and less, as shown by 1H NMR spectra. We first synthesized various nanogels with varying ratios of CH2O:CH2N (methylene proton) in PEG-PEI as shown by 1H NMR spectra and tested their cytotoxicity using a rodent pancreatic adenocarcinoma cell line (Pan 02). We showed that the nanogel PEG-PEI with methylene proton ratio of 4:1 was strongly cytotoxic to Pan 02 cells in vitro, while the nanogel with the methylene proton ratio of 6.8:1 was not toxic. We incorporated a novel anti-cancer drug, 6-(hydroxymethyl)-1,4-anthracenedione (AQ) analogue (AQ10) into nontoxic nanogel PEG-PEI and tested the effect of AQ10 loaded nanogel PEG-PEI (AQ10-nanogel PEG-PEI) and AQ10 dissolved in DMSO on Pan 02 cell growth. The size of this AQ10-nanogel PEG-PEI was characterized using atomic force microscopy (AFM). Our studies showed that the AQ10-nanogel PEG-PEI is readily taken up by Pan 02 cells. Growth attenuation of Pan 02 cells treated with AQ10-nanogel PEG-PEI was three to four times that of cells treated with AQ10 dissolved in DMSO. These results suggest that PEG-PEI, usually used to deliver nucleic acids into cells, can also be used to deliver an insoluble small molecule anticancer drug, AQ10.

Splenic nerve denervation abrogates enhanced splenic cytokine gene expression responses to acute heating, demonstrating that hyperthermia-induced activation of splenic sympathetic nerve discharge (SND) increases splenic cytokine gene expression. Hypothermia alters SND responses; however, the role of the sympathetic nervous system in mediating splenic cytokine gene expression responses to hypothermia is not known. The purpose of the present study was to determine the effect of hypothermia on the relationship between the sympathetic nervous system and splenic cytokine gene expression in anesthetized F344 rats. Gene expression analysis was performed using a microarray containing 112 genes, representing inflammatory cytokines, chemokines, cytokine/chemokine receptors and housekeeping genes. A subset of differentially expressed genes was verified by real-time RT-PCR analysis. Splenic SND was decreased significantly during cooling (core temperature decreased from 38 to 30 degrees C) in splenic-intact rats but remained unchanged in sham-cooled splenic-intact rats (core temperature maintained at 38 degrees C). Hypothermia upregulated the transcripts of several genes, including, chemokine ligands CCL2, CXCL2, CXCL10, and CCL20, and interleukins IL-1alpha, IL-1beta, and IL-6. Gene expression responses to hypothermia were similar for the majority of cytokine genes in splenic-intact and splenic-denervated rats. These results suggest that hypothermia-enhanced splenic cytokine gene expression is independent of splenic SND.

We tested the hypothesis that central angiotensin II (ANG II) administration would activate splenic sympathetic nerve discharge (SND), which in turn would alter splenic cytokine gene expression. Experiments were completed in sinoaortic nerve-lesioned, urethane-chloralose-anesthetized, splenic nerve-intact (splenic-intact) and splenic nerve-lesioned (splenic-denervated) Sprague-Dawley rats. Splenic cytokine gene expression was determined using gene-array and real-time RT-PCR analyses. Splenic SND was significantly increased after intracerebroventricular administration of ANG II (150 ng/kg, 10 μl), but not artificial cerebrospinal fluid (aCSF). Splenic mRNA expression of IL-1β, IL-6, IL-2, and IL-16 genes was increased in ANG II-treated splenic-intact rats compared with aCSF-treated splenic-intact rats. Splenic IL-1β, IL-2, and IL-6 gene expression responses to ANG II were significantly reduced in splenic-denervated compared with splenic-intact rats. Splenic gene expression responses did not differ significantly in ANG II-treated splenic-denervated and aCSF-treated splenic-intact rats. Splenic blood flow responses to intracerebroventricular ANG II administration did not differ between splenic-intact and splenic-denervated rats. These results provide experimental support for the hypothesis that ANG II modulates the immune system through activation of splenic SND, suggesting a novel relation between ANG II, efferent sympathetic nerve outflow, and splenic cytokine gene expression.

Whole body hyperthermia (WBH) has been used in experimental settings as an adjunct to radiochemotherapy for the treatment of various malignant diseases. The therapeutic effect of WBH has been hypothesized to involve activation of the immune system, although the effect of hyperthermia-induced activation of sympathetic nerve discharge (SND) on splenic immune function is not known. We tested the hypothesis that heating-induced splenic sympathoexcitation would alter splenic cytokine gene expression as determined using gene array and real-time RT-PCR analyses. Experiments were performed in splenic-intact and splenic-denervated anesthetized Sprague-Dawley rats (n=32). Splenic SND was increased during heating (internal temperature increased from 38 degrees to 41 degrees C) in splenic-intact rats but remained unchanged in nonheated splenic-intact rats. Splenic interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and growth-regulated oncogene 1 (GRO 1) mRNA expression was higher in heated than in nonheated splenic-intact rats. Splenic IL-1beta, IL-6, and GRO 1 mRNA expression was reduced in heated splenic-denervated compared with heated splenic-intact rats, but did not differ between heated splenic-denervated and nonheated splenic-intact rats. These results support the hypothesis that hyperthermia-induced activation of splenic SND enhances splenic cytokine gene expression.

Animal models are essential to preclinical cancer research and are used clinically to determine optimal treatment regimens, but current xenograft models are limited in their utility, especially due to the lack of a competent immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and, after birth, mice were challenged again with A375 cells to determine their ability to develop tumors. Intravenous injections of A375 cells after tolerization resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. The development of lung neoplasms was dependent on fetal tolerization; non-tolerized mice did not develop tumors after injection. Interestingly, subcutaneous injected cells did not form tumors, but this was shown to be due to an innate, non-adaptive immune response and did not create lasting rejection of tumor cells. This procedure was repeated with several different tumor lines to show the universal nature of the method: BxPC3 (human pancreatic cancer), M21 (human melanoma), HeLa (human cervical cancer), and MDA231 (human breast carcinoma). Citation Format: Matthew T. Basel, Sanjeev Narayanan, Chanran Ganta, Tej B. Shrestha, Marla Pyle, Stefan H. Bossmann, Deryl L. Troyer. Developing a xenograft human tumor model in immunocompetent mice [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 4820. doi:10.1158/1538-7445.AM2017-4820

Hyperthermia is a potent activator of visceral sympathetic nerve discharge (SND), and the functional integrity of the rostral ventral lateral medulla (RVLM) is critically important for sustaining sympathoexcitation at peak hyperthermia. However, RVLM mechanisms mediating SND activation to acute heat stress are not well understood. Because RVLM GABA is tonically inhibitory to sympathetic nerve outflow, it is plausible to hypothesize that disinhibition of RVLM sympathetic neural circuits, via withdrawal of GABAergic tone, may affect SND regulation at peak hyperthermia. The effect of RVLM bicuculline (BIC; GABAA receptor antagonist, 100-200 pmol) microinjections on the level of renal SND in anesthetized rats was determined after internal body temperature (Tc) had been increased to 41.5°C. Temperature-control experiments involved RVLM BIC (100–200 pmol) microinjections, with Tc maintained at 38°C. As expected, acute heating significantly increased renal SND from control levels. Bilateral RVLM BIC microinjections at 41.5°C produced immediate and significant increases in renal SND above heating-induced levels of activation. Bilateral RVLM BIC microinjections at 38°C increased renal SND to similar levels as produced by RVLM BIC microinjections after Tc had been increased to 41.5°C (heating + RVLM BIC). These results demonstrate that a considerable level of RVLM GABAergic inhibition is sustained at peak hyperthermia, an interesting physiological response profile based on the significance of SND activation to cardiovascular regulation during heat stress.

Two 12-year-old cats were diagnosed with chronic kidney disease (CKD) based on physical examination, clinicopathologic data and, in one case, abdominal ultrasound findings. Approximately 1 year after the initial diagnosis of CKD both cats developed renal transitional cell carcinoma (TCC) — bilateral in one cat. Based on post-mortem examination, one cat had no evidence of metastasis and the other had metastasis to the large intestine, heart and lungs. This is the first report of de novo bilateral renal TCC in a cat, as well as the first report of renal TCC developing in cats with previous history of confirmed CKD.

A 4-year 10-month-old, intact female dromedary camel had progressive left carpal joint swelling and lameness for 7 months. Radiographs showed multifocal lytic lesions in the carpal and proximal metacarpal bones. Surgical biopsy of the synovial capsule and carpal bones suggested neoplasia, and the camel was subsequently euthanized. At necropsy, a white to pale pink, firm, multilobulated, soft tissue mass was located on the palmar aspect of the left carpal joint. Two smaller masses were present on the dorsal aspect of the carpal joint. The masses infiltrated all the carpal bones and the proximal region of the metacarpal bone. The joint capsule was diffusely thickened. The articular surfaces of the carpal bones and the metacarpal bone were multifocally eroded. The lungs contained multiple, firm, raised, gray, randomly distributed nodules. The neoplastic cells stained positive for vimentin and S-100. Chondrosarcoma arising from around the carpal joint with infiltration of carpal and metacarpal bones, and pulmonary metastasis, was diagnosed based on the histopathological and immunohistochemical evaluation.

Genetically engineered stem cells efficiently deliver therapeutic proteins to cancer and other sites of inflammation. However, a major advantage would be realized if tumor-trafficking stem cells that have not been genetically modified exhibit an inherent antitumor effect, thus circumventing the necessity of the expression of exogenous genes by the cells. We transplanted Fisher 344 rat–derived mammary adenocarcinoma cells (Mat B III) orthotopically into syngeneic F344 rats with an intact immune system. Rat umbilical cord matrix stem (rUCMS) cells derived from Wharton's jelly were then administered intratumoral (i.t) or i.v. 4 days later. The tumor attenuation effect was significantly evident starting from day 14 in i.v. and i.t. rUCMS cell–transplanted rats compared with sham-transplanted rats. In addition, unmodified rUCMS cell–transplanted rats showed complete regression of tumors to undetectable levels by 34 to 38 days with no evidence of metastasis or recurrence 100 days post–tumor cell inoculation. Dye-loaded rUCMS cells were identified within tumors only 4 days after their i.v. transplantation. In vitro colony assays with rUCMS cells as feeder layers markedly reduced Mat B III colony size and number. Growth attenuation of Mat B III cells exposed to either rUCMS cells directly or to the conditioned medium derived from rUCMS cells was associated with apoptosis indicators, including increased activated caspase-3. In addition, rUCMS cells cocultured with Mat B III cells had a dose-dependent antiproliferative effect on Mat B III cells. These findings suggest that unmodified human UCMS cells could be used for targeted cytotherapy for breast cancer. [Cancer Res 2009;69(5):1815–20]

Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections.

Hypothermia produced by acute cooling prominently alters sympathetic nerve outflow. Skin sympathoexcitatory responses to skin cooling are attenuated in aged compared with young subjects, suggesting that advancing age influences sympathetic nerve responsiveness to hypothermia. However, regulation of skin sympathetic nerve discharge (SND) is only one component of the complex sympathetic nerve response profile to hypothermia. Whether aging alters the responsiveness of sympathetic nerves innervating other targets during acute cooling is not known. In the present study, using multifiber recordings of splenic, renal, and adrenal sympathetic nerve activity, we tested the hypothesis that hypothermia-induced changes in visceral SND would be attenuated in middle-aged and aged compared with young Fischer 344 (F344) rats. Colonic temperature (Tc) was progressively reduced from 38°C to 31°C in young (3 to 6 mo), middle-aged (12 mo), and aged (24 mo) baroreceptor-innervated and sinoaortic-denervated (SAD), urethane-chloralose anesthetized, F344 rats. The following observations were made. 1) Progressive hypothermia significantly ( P < 0.05) reduced splenic, renal, and adrenal SND in young baroreceptor-innervated F344 rats. 2) Reductions in splenic, renal, and adrenal SND to progressive hypothermia were less consistently observed and, when observed, were generally attenuated in baroreceptor-innervated middle-aged and aged compared with young F344 rats. 3) Differences in splenic, renal, and adrenal SND responses to reduced Tc were observed in SAD young, middle-aged, and aged F344 rats, suggesting that age-associated attenuations in SND responses to acute cooling are not the result of age-dependent modifications in arterial baroreflex regulation of SND. These findings demonstrate that advancing chronological age alters the regulation of visceral SND responses to progressive hypothermia, modifications that may contribute to the inability of aged individuals to adequately respond to acute bouts of hypothermia.