Adult C57BL Mice Research Articles

Preclinical Activity of 4-Demethyl-4-cholesteryloxycarbonylpenclomedine in Melanoma.

Temozolomide plays a role in treating melanoma refractory to immunomodulatory and mitogen-activated protein kinase-targeted approaches, but its efficacy is limited. 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate. Its mechanism of action is considered to be via alkylation/adduct formation with N7-guanine. It demonstrated activity in intracranial implanted human glioma and breast cancer xenograft mouse models. The activity of DM-CHOC-PEN in melanoma models was assessed. B-16 melanoma cells were exposed to DM-CHOC-PEN at different concentrations to assess proliferation and survival. B-16 cells were implanted subcutaneously into the flank of adult female C57BL mice which were then were treated with 200 mg/kg DM-CHOC-PEN intraperitoneally daily for 5 days in the setting of palpable subcutaneous tumor. Survival was compared to mice treated with temozolomide or saline. Five mice were treated per group. In vitro, the respective half-maximal inhibitory concentrations of DM-CHOC-PEN and temozolomide were 0.5 and ≥3.0 μg/ml. Floating, heavily melanotic cells formed and these cells were separated, analyzed, and contained 10-90 ng DM-CHOC-PEN per 105 cells. The improvement in survival of mice treated with DM-CHOC-PEN or temozolomide relative to saline controls was 142% and 78%, respectively. Longer survival was seen with DM-CHOC-PEN in a C57BL murine model relative to temozolomide and saline-treated controls, supporting the development of clinical trials assessing the efficacy of DM-CHOC-PEN as treatment for metastatic melanoma.

Abstract 13531: Hydrogen Sulfide Donor, SG1002, Protects Against Hypertension-Induced Exacerbation of Doxorubicin Cardiotoxicity in Female Mice

Background: Dose-dependent declines in left ventricular ejection fraction (LVEF) and increases in heart failure (HF) limit the administration of doxorubicin [DOX] in the treatment of breast cancer, lymphoma, and leukemia. Cancer patients with hypertension (HTN) experience higher rates of developing HF after DOX and perhaps share mechanistic contributors (e.g., oxidative stress and inflammation) to LVEF declines. Hydrogen sulfide (H 2 S) has been shown to stimulate mitochondrial stability and reduce the residual inflammatory risk in the setting of myocardial infarction. Hypothesis: The orally active, slow-releasing H 2 S donor SG1002 attenuates DOX-induced LVEF decline and cardiotoxicity in mice with HTN. Methods: Adult female C57BL mice were randomized to either control diet or SG1002-enriched chow to achieve a dosage of 40 mg/kg/day for the duration of the study. Mice underwent osmotic minipump implantation to deliver angiotensin II (ATII) (1500ng·kg -1 ·min -1 ; N=7/group) for 28 days to cause HTN. Seven days following ATII infusion initiation, DOX was delivered i.p., 4 doses of 5 mg/kg every 3 days (cumulative dose: 20mg/kg). Mice underwent tail-cuff blood pressure measurement and echocardiography at baseline, 12 days (post 2 nd DOX dose) and 28 days post initiation of ATII infusion (Fig A). Results: Twenty-eight-day measures in LVEF were lower in HTN animals receiving DOX relative to those without HTN. SG1002-fed mice demonstrated preserved LVEF following DOX (Fig B). Additionally, SG1002-fed mice exhibited an attenuated hypertensive phenotype following ATII infusion (Fig C). Conclusion: Our results in female mice indicate that HTN worsens DOX-induced cardiomyopathy, which is attenuated with SG1002 treatment. Interestingly, SG1002 also prevented HTN following ATII infusion in female mice. Further studies are warranted to determine the efficacy of the H 2 S donor, SG1002 to prevent DOX-associated LVEF declines in mice with and without HTN.

Inhibition of Metastatic Cancer in Mice via Atopaxar: A Research Protocol

Introduction: When cancer becomes metastatic, tumour cells intravasate out of the primary tumour and spread to other organs, causing about 90% of cancer deaths. One way circulating tumour cells (CTCs) metastasize is by interacting with platelets, resulting in tumour cell-induced platelet aggregation (TCIPA) that shields CTCs from immune attack. Previous studies suggest that tumour cells promote metastasis and induce TCIPA by activating protease-activated receptor-1 (PAR-1) on platelets. Therefore, this study aims to investigate whether administering Atopaxar, a PAR-1 antagonist that has not yet been studied in cancer as other PAR-1 antagonists have, can limit metastasis in mouse models. Methods: We will assess the effectiveness of Atopaxar and a placebo (or control) on adult C57BL mice inoculated with GFP-transfected Lewis lung carcinoma cells. Flow cytometry of blood samples taken 7, 14, and 21-days post-inoculation will be performed to quantify the number of GFP+ cells and activated CD8+ (cytotoxic) T cells in the samples. Results: We expect that the Atopaxar treated mice will have reduced numbers of CTCs and higher numbers of cytotoxic T cells, suggesting that the inhibition of TCIPA via Atopaxar will correlate with reduced shielding of CTCs and metastasis rates. Discussion: These results could provide novel insight into the use of PAR-1 antagonists in confining cancer to its primary site in patients and inhibiting CTCs’ function as a seed for metastases. Conclusion: Since CTCs will usually be present in the blood even after removal of a secondary tumour, limiting metastasis can significantly improve the prognosis and wellbeing of patients.

Evidence of a new hidden neural network into deep fasciae

It is recognized that different fasciae have different type of innervation, but actually nothing is known about the specific innervation of the two types of deep fascia, aponeurotic and epymisial fascia. In this work the aponeurotic thoracolumbar fascia and the epymisial gluteal fascia of seven adult C57-BL mice were analysed by Transmission Electron Microscopy and floating immunohistochemistry with the aim to study the organization of nerve fibers, the presence of nerve corpuscles and the amount of autonomic innervation. The antibodies used were Anti-S100, Anti-Tyrosine Hydroxylase and Anti-PGP, specific for the Schwann cells forming myelin, the sympathetic nerve fibers, and the peripheral nerve fibers, respectively. The results showed that the fascial tissue is pervaded by a rhomboid and dense network of nerves. The innervation was statistically significantly lower in the gluteal fascia (2.78 ± 0.6% of positive area, 140.3 ± 31.6/mm2 branching points, nerves with 3.2 ± 0.6 mm length and 4.9 ± 0.2 µm thickness) with respect to the thoracolumbar fascia (9.01 ± 0.98% of innervated area, 500.9 ± 43.1 branching points/mm2, length of 87.1 ± 1.0 mm, thickness of 5.8 ± 0.2 µm). Both fasciae revealed the same density of autonomic nerve fibers (0.08%). Lastly, corpuscles were not found in thoracolumbar fascia. Based on these results, it is suggested that the two fasciae have different roles in proprioception and pain perception: the free nerve endings inside thoracolumbar fascia may function as proprioceptors, regulating the tensions coming from associated muscles and having a role in nonspecific low back pain, whereas the epymisial fasciae works to coordinate the actions of the various motor units of the underlying muscle.

Comparative Effects of Pyrroloquinoline Quinone (PQQ) and Resveratrol (RSV) on Mitochondriogenesis in Mice

Objectives and Hypothesis Many phytochemicals and biofactors influence mitochondrial function, lipid deposition, and oxidative metabolism. Examples include phytoalexins, flavonoids, and quinones, such as PQQ. Herein, PQQ is compared to RSV (a phytoalexin). Animal tissues retain nM concentrations of PQQ, which promote NAD+ production and sirtuin-related mitochondriogenesis. For example, PQQ promotes mitochondriogenesis in animal models when fed at 0.5 mg PQQ or more/Kg of diet. Such effects elicited by RSV often require 100X or more those amounts. Accordingly, we hypothesize that many of the responses ascribed to RSV may be due to compounds like PQQ or PQQ-derivatives. (cf. Chowanadisai et al., J of Biol Chem 285, 2010:142-52). Methods Two experiments were performed. (Exp. 1) Swiss-derived CD-1 mice express excessive weight gain and fat deposition when exposed to high-fat diets. A goal was to determine if PQQ can attenuate such increases in weight or fat deposition. The mice were maintained in keeping with the FASEB Principles for the Use of Animals in Research and Education. Groups of mice were fed either a low-fat (LFD, 5%) or high-fat (HFD, 20%) casein-based diet with or without PQQ addition at 10 mg/Kg diet for 4 weeks. (Exp.2) Adult C-57BL mice (4-6 months old) were used with or without PQQ addition at 10 mg/Kg diet or RSV at 400 mg/Kg diet. Liver, brown fat, and muscle were examined. The relative numbers of mitochondria were estimated using real-time PCR methods to assess mtDNA/nuclearDNA expression to compare PQQ vs. RSV responses. PQQ in plasma was measured using chemical and mass spectrometric methods. Results (Exp 1) PQQ attenuated the increase in body weight and fat in mice fed the high-fat diet (p<0.05). Mice fed the high-fat diet (-PQQ) weighed 40+/-3 g. Those receiving the high-fat diet (+PQQ) or those containing 5% fat weighed 27 to 32 g. PQQ exposure reduced body fat by 50 % or more**. The relative mtDNA/DNA concentrations in skeletal muscle for CD-1 mice were: 10.8 ± 3.3 (HFD-PQQ) vs. 15.1±1.2 (HFD+PQQ)*, and 18.5 ± 3.0 (LFD-PQQ) vs. 20. 8 ± 3.9 (LFD+PQQ). (Exp.2) PQQ at 1/40th the dietary dose as RSV promoted mtDNA stimulation in liver, brown fat, and skeletal muscle. As a percentage of control values, the mtDNA/DNA ratios, although variable, were increased by 113% and 111% following RSV and PQQ exposures, respectively. For brown fat, the values were 115% and 129% for RSV (NS) and PQQ**, respectively. For skeletal muscle, the values were 104% and 113% for RSV (NS) and PQQ**, respectively. The addition of PQQ to diets markedly enhanced plasma PQQ levels (3±2 pmol PQQ/mL, control; 17±4 pmol PQQ/mL, PQQ suppl.). Of interest, exposure to RSV also increased plasma PQQ (6.5±2 pmol PQQ/mL). [NS=not significant, *=p<0.05, **=0.01] Conclusions and Comments PQQ promotes mitochondriogenesis at lower dietary intakes compared to RSV. For the current observations, 10 mg of PQQ/Kg of diet promoted mtDNA changes equivalent to doses of 400 mg of RSV/ Kg of diet. The data also indicate that PQQ at low levels of exposure normalizes body weight and fat in suspectable mice exposed to a high-fat diet.

Lack of influence of estrogen on myosin phosphorylation and post-tetanic potentiation in muscles from young adult C57BL mice

Estrogen influences myosin phosphorylation and post-tetanic potentiation in murine fast muscle. We tested the hypothesis that this influence is mediated by estrogen effects on skeletal myosin light chain kinase (skMLCK) activity. To this end, extensor digitorum longus muscles from female wildtype and skMLCK-absent (skMLCK-/-) mice were grouped as follows: ovariectomized with estrogen (E+), ovariectomized without estrogen (E-), sham surgery, and intact baseline. At 8 weeks of age, the ovariectomized groups were ovariectomized followed by implantation of either a 0.1 mg 17β-estradiol (E+) or placebo pellet (E-). Two weeks later, muscles were isolated and suspended in vitro (25° C) for determination of regulatory light chain phosphorylation and post-tetanic potentiation. Regulatory light chain phosphorylation was not different across conditions within either genotype although wildtype values were significantly greater than skMLCK-/- values. Consistent with this, the potentiation of concentric twitch force was similar between E+ and E- groups within each genotype but wildtype values were greater than skMLCK-/- values. However, unaltered estradiol levels following ovariectomy, likely due to previously underappreciated confounds of mouse age, development, and growth during estrogen supplementation, prevented direct testing of the hypothesis. Future studies should note the importance of estrous cycles and continuing physiological developments of young adult mice when working with ovarian hormones.

Intravenous injection of AAV-PHP.eB across the blood-brain barrier in the adult mouse for central nervous system gene therapy*

Abstract Objective To verify the neurotypicality of AAV-PHP.eB after tail vein injection in adult mice and its efficiency in crossing the blood-brain barrier (BBB). Methods The rAAV-SYN-GFP plasmid was constructed, and adult C57BL mice were injected with AAV-PHP. eB: SYN-GFP in the tail vein (300 nL, virus titer 3 × 109 vg) and in the prefrontal lobe (50 L, virus titer 5 × 1011 vg). The green fluorescent protein (GFP) signal in the brain was observed at two weeks, while the GFP signal in the peripheral organs was observed at four weeks. Results Two weeks after tail vein injection, GFP expression was observed throughout the brain, especially in the cortex, hippocampus, and geniculate nucleus. No GFP signal was observed or detected by western blotting in the peripheral organs after four weeks. GFP signal was observed mainly at the local site after prefrontal lobe injection. Conclusion AAV-PHP.eB: SYN-GFP can effectively cross the BBB in adult mice. Using a neuron-specific promoter allows exogenous gene expression in neurons; therefore, AAV-PHP.eB can be used as an effective carrier for studying diseases in the central nervous system (CNS).

Chronic Posttraumatic Epilepsy following Neocortical Undercut Lesion in Mice.

Posttraumatic epilepsy (PTE) usually develops in a small percentage of patients of traumatic brain injury after a varying latent period. Modeling this chronic neurological condition in rodents is time consuming and inefficient, which constitutes a significant obstacle in studying its mechanism and discovering novel therapeutics for its prevention and treatment. Partially isolated neocortex, or undercut, is known to induce cortical hyperexcitability and epileptiform activity in vitro, and has been used extensively for studying the neurophysiological mechanism of posttraumatic epileptogenesis. However, whether the undercut lesion in rodents causes chronic epileptic seizures has not been systematically characterized. Here we used a miniature telemetry system to continuously monitor electroencephalography (EEG) in adult C57BL mice for up to 3 months after undercut surgery. We found that 50% of animals developed spontaneous seizures between 16–50 days after injury. The mean seizure duration was 8.9±3.6 seconds, and the average seizure frequency was 0.17±0.17 times per day. There was no progression in seizure frequency and duration over the recording period. Video monitoring revealed behavioral arrests and clonic limb movement during seizure attacks. A pentylenetetrazol (PTZ) test further showed increased seizure susceptibility in the undercut mice. We conclude that undercut lesion in mice is a model of chronic PTE that involves spontaneous epileptic seizures.

Abstract 13498: Reperfusion Therapy With Rapamycin Prevents Myocardial Ischemic Injury, Through Activation of AKT and ERK

Background: The selective inhibitor of mammalian target of rapamycin (mTOR), rapamycin (RAPA), has been shown to exert preconditioning-like cardioprotective effects against ischemia/reperfusion (I/R) injury. Two distinct mTOR complexes (mTORC1 and mTORC2) differentially regulate cardiomyocyte apoptosis and tissue damage following myocardial infarction. We hypothesized that reperfusion therapy with RAPA would reduce myocardial infarct size through differential modulation of mTOR complexes and MAP kinases. Methods and Results: Adult C57BL mice were subjected to 30 min of ischemia via left anterior descending coronary artery ligation followed by reperfusion for 24 hr. RAPA (0.25 mg/kg) or 10% DMSO (volume-matched control) was administered via intra-cardiac injection at the onset of reperfusion. Post I/R survival (90%) and cardiac function (fractional shortening, FS: 26.9±2.6%) were improved in RAPA-treated mice compared to control (survival: 60%, FS:16.7±3.2%). Additionally, RAPA caused significant reduction in myocardial infarct size (Fig. 1A), measured by tetrazolium chloride staining, and apoptosis (Fig. 1B) in peri-infarct regions, assessed by TUNEL staining. Western blot analysis revealed that RAPA restored Akt473 phosphorylation (target of mTORC2), but reduced ribosomal protein S6 phosphorylation (target of mTORC1) following I/R injury (Fig. 1C). The protective effect of RAPA was associated with increased phosphorylation of ERK1/2 and decreased phosphorylation of P38 (Fig. 1D). RAPA also attenuated pro-apoptotic protein Bax, in concert with increased pro-survival Bcl2 to Bax ratio (Fig. 1E). Conclusion: Reperfusion therapy with RAPA protects hearts against I/R injury by selective activation of mTORC2 and ERK with concurrent inhibition of mTOC1 and P38. We propose that RAPA could be a novel treatment strategy to modulate mTOR complexes and MAP kinase signaling for attenuation of reperfusion injury in the heart.

MicroRNA‐21 mediates hydrogen sulfide‐induced protection against ischemia/reperfusion injury in diabetic heart (1080.6)

Background: Hydrogen sulfide (H2S) protects against myocardial ischemia/reperfusion (I/R) and inflammatory injury in mice via induction of microRNA (miR)-21. Since the diabetic heart is non-compliant with most known cardioprotective strategies, we tested whether H2S would also induce miR-21 in the diabetic heart and protect against I/R injury. Methods and Results: Adult db/db mice were treated with vehicle (saline) or the H2S donor, Na2S, (100 µg/kg, iv) at the end of 30 min. of regional ischemia prior to reperfusion for 24 h. After reperfusion, left ventricular function was assessed by echocardiography and infarct size (IS) was measured by TTC staining. Mir-21 expression was assessed by qPCR. Na2S increased miR-21 in db/db mice (5.3-Fold, P<0.005 vs. control) and reduced IS (34±2%) as compared to control db/db mice (68±3%). To test the role of miR-21 in infarct reduction, adult C57BL or miR-21 KO mice were challenged with streptozotocin (STZ; 150 mg/kg, ip) to induce diabetes 4 weeks prior to subjecting them to I/R injury. Na2S reduced IS in STZ-C57BL mice, but not in STZ-miR-21 KO mice (Figure). Moreover, Na2S preserved fractional shortening (FS: 26±3%) post myocardial infarction in STZ-C57BL mice as compared to saline-treated STZ-C57BL mice (16±2%, P<0.05), but not in miR-21 KO-STZ mice (FS: 14±2%). Conclusion: Hydrogen sulfide reduces infarct size and preserves function in the post ischemic diabetic heart through miR-21-dependent pathway. We propose that H2S might be a novel strategy to up-regulate miR-21 for its therapeutic use against I/R injury in diabetics. Grant Funding Source: This research was funded by AHA (10SDG3770011) and VCU PRQF to FNS.

Abstract A264: Response of CNS melanoma to 4-demethyl-4-cholestrylcarbonylpenclomedine (DM-CHOC-PEN) and cell death.

Abstract Introduction: DM-CHOC-PEN is a polychlorinated pyridine cholesteryl carbonate, which has completed a Phase I clinical trial in patients with advanced cancer - IND 68,876 (AACR 1185, 2013). DM-CHOC-PEN produced responses with improved cerebral progression free intervals (CPFI) in patients with CNS melanoma, breast, and lung cancers. CNS melanoma is of especial interest since there is no acceptable therapy for this stage of disease. DM-CHOC-PEN's principle MOA is via alkylation of DNA with N7-guanine, however, in melanoma, there is also generation of reactive oxygen species (ROS) via the DOPA/melanin pathway resulting in melanin encapsulation of melanoma cells and cell death. In mice the drug accumulated in intracerebrally/cerebellar implanted tumors, and not in normal CNS tissue, with no Purkinje cell degeneration; in contrast to drugs such as penclomedine, phencyclidine, ibogaine, etc. that also accumulate in the cerebellum but with cellular destruction. Patients with metastatic CNS cancer treated with DM-CHOC-PEN demonstrated some euphoria, but no CNS toxicity. Methods: B-16 melanoma cells were cultured in RPMI media with 5% FBS and pen/strep at 37oC in a CO2 incubator. Drugs were added to the cells during growth phase and removed after 16 hrs; responses were monitored. Adult female C57BL mice in groups of 5-6 with growing B-16 melanoma were dosed IP daily (75-200 mg/kg) for 5 days with DM-CHOC-PEN or saline and monitored daily until death or moribund and sacrificed. Animals were sacrificed, brain tumor and normal tissue removed and prepared for histology exams. Patients in the Phase I trial (DTI-021) with CNS melanoma were treated with 86.5 mg/m2 IV q 21 days and CNS lesions were monitored with MRI during the Phase I clinical trial (DTI-021) [AACR 1185, 2013]. Results: In vitro, DM-CHOC-PEN had an IC50 0.5 µg/mL against B-16 melanoma cells. Mice bearing B-16 melanoma treated with DM-CHOC-PEN (200 mg/kg/d x 5d, IP) alone vs. saline controls demonstrated %ILS of 142% - supporting the in vitro observations. Patients with CNS melanoma treated with DM-CHOC-PEN demonstrated objective responses and improved CPFI with associated melanin deposits similar to those seen with mice. DM-CHOC-PEN induced ROS via DOPA/DOPA oxidase-melanin deposits and cell death in mice with melanoma. Discussions - DM-CHOC-PEN bound to erythrocytes crosses the BBB and accumulates in CNS melanoma (not normal tissue) with intracellular DOPA-DOPA quinone and melanin formation that interrupts cancer cell metabolism and cellular death via ROS. The drug is not recycled into the systemic circulation. Electronic modeling studies support DM-CHOC-PEN's ability to act as a pyridinium co-factor in the transfer of electrons from DOPA to cytochrome c and the intermediary metabolism pool with generation of ROS. Oxidative stress and ROS generation from a DM-CHOC-PEN/DOPA-melanin pathway has now been confirmed. Cerebellar lesions appear to be especially sensitive. Conclusion: Responses and CPFI for patients with CNS cancers (and no CNS toxicity) seen in the Phase I study have been reported [AACR 1185, 2013]. The principle human toxicity was hepatic. The role for the DOPA/DOPA quinone-melanin pathway with ROS formation will be presented as a key MOA for the drug, with emphasis on cerebellar melanoma lesions. Supported in part by: NCI/ SBIR grants R43/44CA85021 and R43/44CA132257. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A264. Citation Format: Lee Roy Morgan, Edmund Benes, Andrew H. Rodgers, Roy S. Weiner, Marcus L. Ware, Philip Friedlander, Yvonne Saenger, Craig Gordon, Tallat Mahmood, Branko Jursic, Gerard Bastian. Response of CNS melanoma to 4-demethyl-4-cholestrylcarbonylpenclomedine (DM-CHOC-PEN) and cell death. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A264.

Abstract B219: 4-Demethyl-4-cholesteryloxycarbonylpenclomedine (DM-CHOC-PEN) and 4-hydroperoxyifosfamide (HOOI) as binary therapy for melanoma.

Abstract Introduction: 4-Demethyl-4-cholesteryloxycarbonyl-penclomedine (DM-CHOC-PEN) is a polychlorinated pyridine cholesteryl carbonate, which is in Phase I clinical trials in patients with advanced cancer - IND 68,876. DM-CHOC-PEN is active vs. intracranially (IC) implanted human xenograft models - U251 and D54 glioblastoma and MX-1 breast cancer and recently found to be active vs. B-16 melanoma - 142% ILS. DM-CHOC-PEN's MOA is via alkylation of DNA @ N7 - guanine, as well as converting melanoma cells into a melanotic Go phase with cell death. Not all cells were converted into Go phase and senescence, thus the interest in designing a binary drug approach for DM-CHOC-PEN, as an improvement in treatment for melanoma. A number of agents are cytotoxic vs. B-16 cells in vitro and in vivo; however, 4-hydroperoxyifosfamide (HOOI), which is converted to isophosphoramide mustard (IPM) in vivo, demonstrated the best %ILS. The latter drug has been chosen as a companion with DM-CHOC-PEN in the present binary drug study vs. B-16 mouse melanoma. Methods: B-16 melanoma cells were cultured using RPMI media with 10% FBS and pen/strep @ 37° C in a CO2 incubator. Drugs were added to the cells in a growth phase and removed after 8–12 h. Adult female C57BL mice in groups of 5–6 mice were implanted subcutaneously (SC) with B-16 mouse melanoma (106 cells) and when SC nodules were palpable the mice were dosed IP daily (200 mg/kg) for 5-days with DM-CHOC-PEN followed by HOOI administered IP @ varying doses and daily/weekly schedules and monitored daily until death. Mice with SC B-16 melanoma were dosed with single agents - DM-CHOC-PEN, HOOI, cis-platinum and temozolamide, which were used as controls. Tumor tissue was extracted with dichloromethane, and assayed per HPLC and NMR. Results: In vitro, DM-CHOC-PEN and HOOI, as single agents, had IC50 of 0.5 and 0.8 μg/mL vs. B-16 melanoma cells, resp. Mice bearing SC B-16 melanoma treated with DM-CHOC-PEN (200 mg/kg/d × 5d, IP) alone vs. saline controls demonstrated %ILS of 142%; thus supporting the in vitro observations. In vivo in the B-16 melanoma model, cis-platinum as a single agent had a %ILS = 0%, but for HOOI it was 85%. In 2-drug studies, DM-CHOC-PEN plus cis-platinum together (in theoretical therapeutic ranges) were too toxic in combination, however, the %ILS for DM-CHOC-PEN plus HOOI was 173%. Tumor tissue was removed within 2-days of treating mice with DM-CHOC-PEN, extracted and revealed 75 μg/g tumor tissue of DM-PEN, a metabolite. Discussion: HOOI is a S-phase alkylating agent that is an appropriate 2nd agent to kill cells escaping from the DM-CHOC-PEN - induced Go phase induction. To date, the best treatment regimen was DM-CHOC-PEN (200 mg/kg/d × 5d) followed by HOOI (90 mg/d × 3d) - %ILS = 173. The finding that melanoma tissue extracts resulted in μg of DM-PEN (the metabolite) is in agreement with the pharmacokinetic findings observed for DM-CHOC-PEN in rats and humans, which will be reviewed. The binary drug combination will be reviewed with the FDA. Supported in part by: NCI SBIR grants - R43/44CA85021. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B219.

Underdeveloped Extraocular Muscles in the Naked Mole‐Rat (Heterocephalus glaber)

The extraocular muscles (EOM), the effector arm of the ocular motor system, have a unique embryological origin and phenotype. The naked mole-rat (NMR) is a subterranean rodent with an underdeveloped visual system. It has not been established if their ocular motor system is also less developed. The NMR is an ideal model to examine the potential codependence of oculomotor and visual system development and evolution. Our goal was to compare the structural features of NMR EOMs to those of the mouse, a similar sized rodent with a fully developed visual system. Perfusion-fixed whole orbits and EOMs were dissected from adult NMR and C57BL mice and examined by light and electron microscopy. NMR orbital anatomy showed smaller EOMs in roughly the same distribution around the eye as in mouse and surrounded by a very small Harderian gland. The NMR EOMs did not appear to have the two-layer fiber distribution seen in mouse EOMs; fibers were also significantly smaller (112.3 +/- 46.2 vs. 550.7 +/- 226 sq microm in mouse EOMs, *P < 0.05). Myofibrillar density was less in NMR EOMs, and triad and other membranous structures were rudimentary. Finally, mitochondrial volume density was significantly less in NMR EOMs than in mouse EOM (4.5% +/- 1.9 vs. 21.2% +/- 11.6, respectively, *P < 0.05). These results demonstrate that NMR EOMs are smaller and less organized than those in the mouse. The "simpler" EOM organization and structure in NMR may be explained by the poor visual ability of these rodents, initially demonstrated by their primitive visual system.

Underdeveloped extraocular muscles in the naked mole rat (Heterocephalus glaber)

The extraocular muscles (EOM) are the final effector arm of the ocular motor system. These small muscles have a unique embryological origin and phenotype. Their diversity is highlighted by their resistance to some neuromuscular disorders (muscular dystrophy, ALS) and increased susceptibility to others (myasthenia gravis and mitochondrial myophathies). The naked mole rat (NMR) is a subterranean rodent with a reduced visual system; it is an ideal model to examine the potential codependence of full EOM and visual system phenotype development and evolution. Our goal was to compare the structural features of NMR EOMs to those of a similar sized rodent with a fully developed visual system. Whole orbits and EOMs were dissected from perfusion-fixed adult NMR and C57BL mice and examined by light and electron microscopy. NMR orbital anatomy showed smaller EOMs in roughly the same distribution around the eye as in mouse and surrounded by a very small Harderian gland. The NMR EOMs did not appear to have the two-layer fiber distribution (orbital and global) seen in mouse EOMs; fibers were also significantly smaller (118 vs. 551 sq um in mouse EOMs). Myofibrillar density was less in NMR EOMs, and triad and other membranous structures were rudimentary. Finally, mitochondrial volume density was significantly less in NMR EOMs than in mouse EOM (4.5% vs. 21.2% respectively). These results demonstrate that NMR EOMs are smaller and less well organized than those in a rodent with a fully developed visual system. It is likely that at least some aspects of EOM organization depend on visual experience.

Inactivation of PU.1 in adult mice leads to the development of myeloid leukemia

Genetically primed adult C57BL mice were deleted of exon 5 of the gene encoding the transcription factor PU.1 by IFN activation of Cre recombinase. After a 13-week delay, conditionally deleted (PU.1(-/-)) mice began dying of myeloid leukemia, and 95% of the mice surviving from early postinduction death developed transplantable myeloid leukemia whose cells were deleted of PU.1 and uniformly Gr-1 positive. The leukemic cells formed autonomous colonies in semisolid culture with varying clonal efficiency, but colony formation was enhanced by IL-3 and sometimes by granulocyte-macrophage colony-stimulating factor. Nine of 13 tumors analyzed had developed a capacity for autocrine IL-3 or granulocyte-macrophage colony-stimulating factor production, and there was evidence of rearrangement of the IL-3 gene. Acquisition of autocrine growth-factor production and autonomous growth appeared to be major events in the transformation of conditionally deleted PU.1(-/-) cells to fully developed myeloid leukemic populations.

Tissue localisation of tetra- and pentabromodiphenyl ether congeners (BDE-47, -85 and -99) in perinatal and adult C57BL mice

The distribution of polybrominated diphenyl ether (PBDE) congeners was followed in C57BL mice. The animals were subjected to whole-body autoradiography using 14C-labelled 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), 2,2′,3,4,4′-pentabromodiphenyl ether (BDE-85) and 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99). Labelled BDE-85 and -99 were also used in quantitative studies on milk transfer and tissue concentrations during the neonatal period (12–15 days post partum), by use of liquid scintillation technique. The results show that in adult mice the studied PBDEs were effectively taken up, retained in fatty tissues and concentrated in some specific organs, i.e. the liver, adrenal cortex, ovary, lung and (initially) the brain. At longer post-injection time, the concentration in most tissues was considerably lower, and radioactivity was mainly found in fat depots and the liver. No significant difference in distribution between the three studied congeners was observed. Following maternal exposure, the foetal uptake was limited. On the other hand, during lactation a considerable fraction of the dose (about 20% of the studied penta-BDEs) given to the dam was transferred to the offspring. As in several cases the presently observed organ accumulation corresponds with earlier reports on PBDE effects in the same organs, the present results should be taken into consideration in the risk assessment of this compound group.

Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague–Dawley rat

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague–Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400–900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague–Dawley rats, doses of 1×500 mg or 2×300 mg kanamycin base/kg body weight/day×14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/ Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

Drs Veninga, Morse and Wieringa reply:

Drs Veninga, Morse and Wieringa reply: Sir, The comments made by Dr Sundberg are very much appreciated, and we thank him for providing copies of the references. It is our opinion, however, that the references cited by Dr Sundberg convincingly support the findings we reported on pigmented spleens seen in 150/0of our C57BL mice (Veninga et al., 1989), rather than refute them. For the sake of clarity, the references cited in our reply will be those provided by Dr Sundberg. The observed pigmentation in our study was proved to be primarily haemosiderin and not melanin because: (a) Macroscopically, the pigment colours up to twothirds of the spleen uniformly, giving the affected part a black appearance. It is not present in localized spots as has been described for melanin (Weissmann, 1967). It is of interest to note that the black part is maintained in spleens of animals infected with 106 lymphosarcoma ceIls,causing an exponentially growing splenic tumour which loosens the tissue in one week. It enabled us to obtain isolated macrophages filled with pigment from 2 such spleens. (b) Microscopically, the pigment is yellow-brown which is characteristic for haemosiderin (Frith & Ward, 1988). (c) The pigment specificaIly stains with prussian blue which proves haemosiderin and absolutely excludes melanin (Frith & Ward, 1988; Sundberg, 1989), with the exception of a small number of very dark granules which are possibly the melanin Dr Sundberg refers to. (d) Unlike melanin, the pigment is present in macrophages (Frith & Ward, 1988; Veninga et at., 1989), although some loose granules were observed outside the cells. These elements have not the characteristic elongated appearance of melanin spots (Frith & Ward, 1988; Sundberg, 1989). (e) The EM picture in our article shows pigment granules surrounded by a membrane, typical for haemosiderin. We agree that such membrane is less clear for a second set of granules in the same cell. Nevertheless, their arrangement is very similar and therefore suggestive of a coherent structure. Both granule clusters are enclosed in a macrophage, the typical cell type containing haemosiderin (Frith & Ward, 1988). We realize that our article obviously was not completely clear. As indicated, most of the mice we worked with were 3 months of age; after publication of the article we also discovered pigmented spleens in 2-month-old animals, showing that this is not strictly an age-onset condition. Although we would be the last to deny the possibility that a few melanin particles (such as the particles not staining with prussian blue) were present in the spleens of our mice, we prefer our original interpretation (Veninga et al., 1989). In conclusion, we maintain that the pigmented apex of the spleens of 15% of our young adult C57BL mice contain concentrations principally of haemosiderin, not melanin a disorder characteristic for haemosiderosis. Tjeerd Veninga Howard Morse Andre Wieringa

Different mechanisms of protection by monoclonal and polyclonal antibodies during the course of herpes simplex virus infection.

Young adult C57BL mice intravaginally inoculated with herpes simplex virus type 1 and passively immunized with a monoclonal antibody directed against herpes simplex virus type 1 glycoprotein gB were shown to be more effectively protected against infection as compared with mice treated with polyclonal immune serum. In contrast to polyclonal antiserum, the monoclonal antibody markedly restricted viral multiplication in the infected mucous membranes. Consequently, skin lesions were completely prevented, and the extent of ganglionic infection was significantly reduced. The mechanism by which a monoclonal antibody, specific to glycoprotein gC, effected protection also differed from that of the hyperimmune serum, since premature latency was not induced. The data provide strong evidence that the mechanisms of protection mediated by antibodies depend on their epitope specificity. The inhibition of active antibody response after passive immunization was inducible by polyclonal antibody only, not by monoclonal antibodies.

Sexual dimorphism in amino acid compositions of mouse prolactin

Sexual dimorphism was found in amino acid compositions and immunogenecity of variant types of prolactin (PRL) purified from the pituitary gland of normal adult C57BL mice by a high performance liquid chromatography. From the pituitary gland of female mice, three female variant types of PRL were isolated, whereas from the pituitary gland of male mice, two male variant types of PRL (M1-PRL and M3-PRL) and a female variant type of PRL (M2-PRL) were obtained. The amino acid composition of M3-PRL was different from any of female variant types which were very similar to each other and contained less varine, isoleucine, leucine, tyrosine and lysine but more alanine than the latter. Immunoreactivity of any of female variant types of PRL against anti-PRL serum was 100%, whereas that of M1-PRL was as much as 85% and that of M3-PRL was nearly undetectable.