Severe Body Weight Loss Research Articles

A Mouse Model of Shigellosis by Intraperitoneal Infection

In human and nonhuman primates, Shigella spp. cause bacillary dysentery by invading colon epithelium and promoting a strong inflammatory response; however, adult mice are resistant to oral Shigella infection. In this study, intraperitoneal challenge with virulent S. flexneri 2a (YSH6000) resulted in diarrhea and severe body weight loss in adult B6 mice. Of note, virulent S. flexneri 2a could invade and colonize not only systemic tissues but also the serosa and lamina propria region of the large intestine. In addition, epithelial shedding, barrier integrity, and goblet cell hyperplasia were found in the large intestine by 24 hours post-intraperitoneal Shigella infection. Of note, predominant expression of proinflammatory cytokines and chemokines were found in the large intestine after intraperitoneal challenge. Monocytes played a critical role in attenuating diarrhea and in providing protective efficacy against intraperitoneal Shigella infection. Most importantly, mice prevaccinated with attenuated S. flexneri 2a (SC602) strain were protected against intraperitoneal challenge with YSH6000. When taken together, these findings show that intraperitoneal challenge with virulent S. flexneri 2a can provoke bacillary dysentery and severe pathogenesis in adult mice. This model may be helpful for understanding the induction mechanism of bacillary dysentery and for evaluating Shigella vaccine candidates.

Abstract 5613: Enhanced treatment of lung metastasis of triple negative breast cancer by doxorubicin-mitomycin C co-loaded polymer lipid nanoparticles.

Abstract Background: Treatment of triple negative breast cancer (TNBC) is a big challenge due to its aggressiveness, metastases, lack of therapeutic target and receptor proteins. Our laboratory has developed doxorubicin (Dox) -mitomycin C (MMC) co-loaded stealth polymer lipid hybrid nanoparticles (DMsPLN) and demonstrated their anti-cancer synergy in vitro and high efficacy in vivo. The purpose of this study is to evaluate the biodistribution, in vivo efficacy and safety of DMsPLN in a lung metastatic human TNBC model. Method: Lung metastasis of breast tumor was established using MDA-MB 231-luc-D3H2LN, a luciferase expressing cell line that was derived from spontaneous lymph node metastasis. The cells were injected via tail vein to develop lung metastasis in SCID mice. The biodistribution and tumor accumulation of the nanoparticles were examined by whole animal optical imaging using near infrared fluorescence labeled nanoparticles. The efficacy and systemic toxicity of DMsPLN were evaluated against clinically used doxorubicin (Dox). DMsPLN and free Dox solution were administered intravenously at various equivalent Dox doses. The tumor burden was monitored weekly by bioluminescence imaging. The size and number of tumor nodules in the lungs were examined at different time points and the systemic toxicity was monitored by repeated measurement of body weight. Results: A lung metastatic breast tumor model was successfully developed using the MDA-MB 231-luc-D3H2LN which allowed for non-destructive monitoring of tumor growth and metastases using bioluminescence imaging. Whole animal imaging demonstrated the accumulation of the fluorescent nanoparticles in the lung metastatic site. Treatment with free Dox (10 mg/kg) resulted in severe total body weight loss over 20% which is ruled as a toxic endpoint. However, the DMsPLN (10 mg/kg equivalent Dox dose) group did not show any systemic toxicity. Treatment with DMsPLN at various Dox doses resulted in a reduction in tumor burden compared to the saline and Dox-treated groups. Citation Format: Preethy Prasad, Ping Cai, Andrew Rauth, Xiao Wu. Enhanced treatment of lung metastasis of triple negative breast cancer by doxorubicin-mitomycin C co-loaded polymer lipid nanoparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5613. doi:10.1158/1538-7445.AM2013-5613 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Neuroprotective effects of lactate in brain ischemia: Dependence on anesthetic drugs

Lactate is a major energy source for the brain, especially when glucose is not available in sufficient amounts. In the present study, we administered sodium l-lactate (250mg/kg) to mice before or after middle cerebral artery occlusion (MCAO) to test whether lactate can be neuroprotective in brain ischemia. Permanent ischemia for 24h caused a large hemispheric lesion and a severe loss of body weight. Administration of lactate shortly (15–30min) before MCAO strongly reduced cell death and weight loss, but only when isoflurane was used for anesthesia. Under pentobarbital anesthesia, lactate was inactive. After transient ischemia, when isoflurane or ketamine–xylazine were used as anesthetic drugs, lactate was effective when given immediately after reperfusion. In separate experiments, we found that plasma lactate levels are also strongly influenced by anesthetic drugs. Thus, isoflurane anesthesia as well as lactate administration caused strongly increased plasma levels of lactate whereas pentobarbital anesthesia significantly reduced plasma lactate. We conclude that exogenous lactate is neuroprotective in an in vivo-model of brain ischemia, but that its action is strongly influenced by the type of anesthetic agent used.

Gait Impairment in a Rat Model of Focal Cerebral Ischemia

The availability of proper tests for gait evaluation following cerebral ischemia in rats has been limited. The automated, quantitative CatWalk system, which was initially designed to measure gait in models of spinal cord injury, neuropathic pain, and peripheral nerve injury, is said to be a useful tool for the study of motor impairment in stroke animals. Here we report our experiences of using CatWalk XT with rats subjected to transient middle cerebral artery occlusion (MCAO), during their six-week followup. Large corticostriatal infarct was confirmed by MRI in all MCAO rats, which was associated with severe sensorimotor impairment. In contrast, the gait impairment was at most mild, which is consistent with seemingly normal locomotion of MCAO rats. Many of the gait parameters were affected by body weight, walking speed, and motivation despite the use of a goal box. In addition, MCAO rats showed bilateral compensation, which was developed to stabilize proper locomotion. All of these interferences may confound the data interpretation. Taken together, the translational applicability of CatWalk XT in evaluating motor impairment and treatment efficacy remains to be limited at least in rats with severe corticostriatal infarct and loss of body weight.

Effects of selenium supplementation and chronic inflammation on bone microarchitecture and strength in mice

Timed‐pregnant C57BL6 mice were fed a Torula yeast selenium‐depletion diet from the final days of gestation through lactation. At 23 days of age, pups were weaned and randomly assigned to the depletion diet or to diets supplemented with 0.2, 2 or 4 mg/kg diet of Se added as sodium selenate for 14 weeks. At 96–98 days of age mice were randomly assigned to placebo or to lipopolysaccharide (E. coli Serotype 0127:B8) treatment to produce chronic inflammation. Time release pellets (0 or 0.1 μg/g body weight/d) were implanted subcutaneously. Body weight was reduced by LPS 14 days after implantation but differences were no longer significant by 28 days. Bone microarchitecture was evaluated in tibia and 4th lumbar vertebra (L4) and force to compress was estimated on trabecular cores using finite element analysis (Scanco μCT40). Bone volume/total volume, connectivity density, and trabecular number were significantly reduced by LPS treatment while trabecular separation was increased by LPS treatment in both bones. In spine significant interactions affected size‐independent stiffness and von Mises stresses. Overall, chronic inflammation at a level that does not produce severe body weight loss still significantly impaired measures of bone microarchitecture and strength. (Supported by Oklahoma Center for Advancement of Science and Technology).

Induction of Heterosubtypic Cross-Protection against Influenza by a Whole Inactivated Virus Vaccine: The Role of Viral Membrane Fusion Activity

BackgroundThe inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV) vaccine, that can target conserved internal antigens such as the nucleoprotein (NP) and/or matrix protein (M1) need to be explored.Methodology/Principal FindingsIn the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs), protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA) severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL) preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge.Conclusion/SignificanceThe results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane fusion activity and full immunogenicity of the vaccine.

Rapid-Onset Hyponatremia Induced by Duloxetine in a Middle-Aged Male with Depression and Somatic Symptoms

Duloxetine is a relatively balanced selective serotonin and noradrenaline reuptake inhibitor. We report a case of hyponatremia induced by duloxetine developed rapidly after starting the medication in a middle-aged male with multiple somatic symptoms and depression. Two days after discontinuation of duloxetine and management with hypertonic saline as well as fluid restriction, the serum sodium level normalized. The patient had two risk factors for developing hyponatremia, such as severe body weight loss and pneumonia. Therefore, when treating patients with depression and somatic symptoms, especially with risk factors for developing hyponatremia, close monitoring for clinical and laboratory evidence of hyponatremia may be essential.

Immunological characteristics and response to lipopolysaccharide of mouse lines selectively bred with natural and acquired immunities

Genetic improvement of resistance to infectious diseases is a challenging goal in animal breeding. Infection resistance involves multiple immunological characteristics, including natural and acquired immunity. In the present study, we developed an experimental model based on genetic selection, to improve immunological phenotypes. We selectively established three mouse lines based on phagocytic activity, antibody production and the combination of these two phenotypes. We analyzed the immunological characteristics of these lines using a lipopolysaccharide (LPS), which is one of the main components of Gram-negative bacteria. An intense immunological reaction was induced in each of the three mouse lines. Severe loss of body weight and liver damage were observed, and a high level of cytokine messenger RNA was detected in the liver tissue. The mouse line established using a combination of the two selection standards showed unique characteristics relative to the mouse lines selected on the basis of a single phenotype. Our results indicate that genetic selection and breeding is effective, even for immunological phenotypes with a relatively low heritability. Thus, it may be possible to improve resistance to infectious diseases by means of genetic selection.

Toll-Like Receptors 2 and 4 Regulate the Frequency of IFNγ-Producing CD4+ T-Cells during Pulmonary Infection with Chlamydia pneumoniae

TLR2 and TLR4 are crucial for recognition of Chlamydia pneumoniae in vivo, since infected TLR2/4 double-deficient mice are unable to control the infection as evidenced by severe loss of body weight and progressive lethal pneumonia. Unexpectedly, these mice display higher pulmonary levels of the protective cytokine IFNγ than wild type mice. We show here, that antigen-specific CD4+ T-cells are responsible for the observed IFNγ-secretion in vivo and their frequency is higher in TLR2/4 double-deficient than in wild type mice. The capacity of TLR2/4 double-deficient dendritic cells to re-stimulate CD4+ T-cells did not differ from wild type dendritic cells. However, the frequency of CD4+CD25+Foxp3+ T-cells was considerably higher in wild type compared to TLR2/4 double-deficient mice and was inversely related to the number of IFNγ-secreting CD4+ effector T-cells. Despite increased IFNγ-levels, at least one IFNγ-mediated response, protective NO-secretion, could not be induced in the absence of TLR2 and 4. In summary, CD4+CD25+Foxp3+ regulatory T-cells fail to expand in the absence of TLR2 and TLR4 during pulmonary infection with C. pneumoniae, which in turn enhances the frequency of CD4+IFNγ+ effector T-cells. Failure of IFNγ to induce NO in TLR2/4 double-deficient cells represents one possible mechanism why TLR2/4 double-deficient mice are unable to control pneumonia caused by C. pneumoniae and succumb to the infection.

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in β1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5-/- mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5-/- mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that β1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.

A Recombinant Influenza A Virus Expressing Domain III of West Nile Virus Induces Protective Immune Responses against Influenza and West Nile Virus

West Nile virus (WNV) continues to circulate in the USA and forms a threat to the rest of the Western hemisphere. Since methods for the treatment of WNV infections are not available, there is a need for the development of safe and effective vaccines. Here, we describe the construction of a recombinant influenza virus expressing domain III of the WNV glycoprotein E (Flu-NA-DIII) and its evaluation as a WNV vaccine candidate in a mouse model. FLU-NA-DIII-vaccinated mice were protected from severe body weight loss and mortality caused by WNV infection, whereas control mice succumbed to the infection. In addition, it was shown that one subcutaneous immunization with 105 TCID50 Flu-NA-DIII provided 100% protection against challenge. Adoptive transfer experiments demonstrated that protection was mediated by antibodies and CD4+T cells. Furthermore, mice vaccinated with FLU-NA-DIII developed protective influenza virus-specific antibody titers. It was concluded that this vector system might be an attractive platform for the development of bivalent WNV-influenza vaccines.

Abstract 5423: New diazonamide analogs demonstrate remarkably larger therapeutic window compared to taxanes and vinca alkaloids in rodents

Abstract Diazonamide A is a natural product originally isolated from a marine organism. It potently induces G2/M cell cycle arrest. Its molecular mode of action is not fully understood but its potential as a cancer therapeutic is high. To streamline our diazonamide synthesis and to define a minimal pharmacophore, we prepared a series of truncated structures. Its simplified structure and shortened synthesis allowed for extensive structure activity relationship analyses. Several modifications markedly improved activity. Among them, JP2295 and JP2362 were studied in details. Both compounds were highly efficacious in a wide spectrum of tumors from different origin both in vitro and in vivo. They were >20 fold more potent than AB5, a closely related analog of the natural product, in a MIA Paca-2 pancreatic cancer xenograft model. At dose 10-fold higher than the efficacious dose, JP2295 or JP2362 did not cause any apparent toxicity. Three out of 7 mice were completely free of tumor even 6 months after the initial treatment. In the same model, taxanes, i.e. paclitaxel and docetaxel, and vinca alkaloids, i.e. vinorelbine, vinblastine, and vincristine, were either less efficacious or causing severe body-weight loss, even animal death. Similar effects were observed in other xenograft models. Safety studies were further carried out in NIH Swiss mice and Sprague Dawley rats with similar results. In summary, JP2295 and JP2362 demonstrated over 20-fold bigger therapeutic window than taxanes and vinca alkaloids in rodents. Such a large therapeutic window is unique for a small molecule anti-mitotic and suggests further development is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2011-5423

Extracellular matrix protein lumican regulates inflammation in a mouse model of colitis

Abnormal innate immune response contributes to inflammatory bowel disease (IBD) and experimental mouse colitis. Colitis studies have focused primarily on key regulators of innate immunity, like pathogen recognition receptors and cytoplasmic mediators. Extracellular matrix (ECM) proteins are emerging as modulators of inflammatory responses by virtue of their interactions with pathogen-associated molecular patterns (PAMPs), cytokines, growth factors, receptors, and ECM fragments that mimic pathogens or cytokines. The ECM proteins have not been investigated in IBD at great depth from this standpoint. We have shown previously that the ECM protein lumican modulates host sensing of bacterial lipopolysaccharides (LPS) by Toll-like receptor (TLR) 4, and neutrophil chemotaxis via integrins. Here we investigated the role of lumican in the development of colitis mediated by intrarectal administration of the hapten 2-4-5, trinitrobenzene sulfonic acid (TNBS) in Lum(+/+) and Lum(-/-) mice. The TNBS treated Lum(+/+) mouse colons showed marked increases in CXCL1, tumor necrosis factor alpha (TNF-α), and neutrophil infiltration, whereas these responses were significantly dampened in the Lum(-/-) mice. The nuclear factor kappa B (NF-κB) transcription factor, known to regulate inflammatory genes, showed a robust increase after TNBS treatment in Lum(+/+) but not in Lum(-/-) colons. Also, nuclear translocation of NF-κB was delayed in LPS stimulated Lum(-/-) primary peritoneal macrophages. The Lum(-/-) mice have low innate immune and inflammatory responses, but more severe body weight loss and tissue damage, a phenomenon seen in the innate immune impaired Tlr4(-/-) and MyD88(-/-) mice. Therefore, lumican promotes intestinal homeostasis by aiding innate immune and inflammatory responses that are beneficial in the early stages of colitis.

Metronomic S-1 Chemotherapy and Vandetanib: An Efficacious and Nontoxic Treatment for Hepatocellular Carcinoma

Metronomic chemotherapy involves frequent, regular administration of cytotoxic drugs at nontoxic doses, usually without prolonged breaks. We investigated the therapeutic efficacies of metronomic S-1, an oral 5-fluorouracil prodrug, and vandetanib, an epidermal growth factor receptor and vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, in models of hepatocellular carcinoma (HCC). We compared anti-HCC effects and toxicity in the six treatment groups: control (untreated), maximum tolerated dose (MTD) S-1, metronomic S-1, vandetanib, MTD S-1 with vandetanib, and metronomic S-1 with vandetanib. Tumor microvessel density (MVD) and tumor apoptosis were evaluated by immunohistochemistry. The expression of VEGF and thrombospondin-1, an endogenous inhibitor of angiogenesis, was analyzed by Western blot. Metronomic S-1 significantly inhibited tumor growth, which was enhanced by combination with vandetanib. With respect to toxicities, MTD S-1 caused severe body weight loss and myelosuppression, whereas metronomic S-1 did not cause any overt toxicities. Moreover, metronomic S-1 or metronomic S-1 with vandetanib prolonged survival, the latter treatment providing the greatest benefit. Metronomic S-1 and metronomic S-1 with vandetanib decreased MVDs and increased apoptosis in tumor tissues. The expression of VEGF in tumor tissues was upregulated by vandetanib and metronomic S-1 with vandetanib, whereas the expression of thrombospondin-1 was upregulated by metronomic S-1 and metronomic S-1 with vandetanib. Metronomic S-1 with an antiangiogenic agent seems to be an effective and safe therapeutic strategy for HCC.

Middle-preserving pancreatectomy for advanced transverse colon cancer invading the duodenun and non-functioning endocrine tumor in the pancreatic tail.

A 73-year-old female was referred to our hospital with a diagnosis of advanced transverse colon cancer with severe anemia and body weight loss. Preoperative evaluations, including colonoscopy, gastroduodenoscopy, and computed tomography, revealed not only a transverse colon cancer massively invading the duodenum, but also a non-functioning endocrine tumor in the pancreatic tail. We performed middle-preserving pancreatectomy (MPP) with right hemicolectomy for these tumors with a curative intent. After the resection, about 6cm of the body of the pancreas was preserved, and signs of diabetes mellitus have not appeared. The postoperative course was complicated by a grade B pancreatic fistula, but this was successfully treated with conservative management. After a 33-day hospital stay, the patient returned to daily life without signs of pancreatic exocrine insufficiency. Although the long-term follow-up of the patient is indispensable, in this case, MPP might be able to lead to the curative resection of transverse colon cancer massively invading the duodenum and non-functioning endocrine tumor in the pancreatic tail with preservation of pancreatic function.

Improved protection against avian influenza H5N1 virus by a single vaccination with virus-like particles in skin using microneedles

To develop a more effective vaccination method against H5N1 virus, we investigated the immunogenicity and protective efficacy after skin vaccination using microneedles coated with influenza virus-like particles containing hemagglutinin derived from A/Vietnam/1203/04 H5N1 virus (H5 VLPs). A single microneedle vaccination of mice with H5 VLPs induced increased levels of antibodies and provided complete protection against lethal challenge without apparent disease symptoms. In contrast, intramuscular injection with the same vaccine dose showed low levels of antibodies and provided only partial protection accompanied by severe body weight loss. Post-challenge analysis suggested that improved protection was associated with lower lung viral titers and enhanced generation of recall antibody secreting cells by microneedle vaccination. Thus, this study provides evidence that skin delivery of H5 VLP vaccines using microneedles designed for self-administration induces improved protection compared to conventional intramuscular immunization.

Protective role of the leukotriene B4receptor BLT2 in murine inflammatory colitis

BLT2 is a low-affinity leukotriene B(4) (LTB(4)) receptor that is activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB(4). Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. To clarify the role of BLT receptors in intestinal inflammation, we assessed susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice lacking either BLT1 or BLT2. BLT2(-/-) mice exhibited increased sensitivity to DSS as compared to wild-type and BLT1(-/-) mice, with more severe body weight loss and inflammation. Expression of inflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-1β, and IL-6, chemokines such as CXC chemokine ligand 9 (CXCL9) and C-C motif chemokine 19 (CCL19), and metalloproteinases was highly up-regulated in the colons of DSS-treated BLT2(-/-) mice, and there was an enhanced accumulation of activated macrophages. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also markedly accelerated in the crypts of DSS-treated BLT2(-/-) mice. Madin-Darby canine kidney II (MDCKII) cells transfected with BLT2 exhibited enhanced barrier function as measured by transepithelial electrical resistance (TER) and FITC-dextran leakage through MDCK monolayers. Thus, BLT2 is expressed in colon cryptic cells and appears to protect against DSS-induced colitis, possibly by enhancing barrier function in epithelial cells of the colon. These novel results suggest a direct anti-inflammatory role of BLT2 that is distinct from the proinflammatory roles of BLT1.

Abstract 1389: Low dose metronomic chemotherapy of S-1 and Vandetanib produces nontoxically good therapeutic results for hepatocellular carcinoma (HCC)

Abstract [Background and aim] A conventional chemotherapy for cancer, which is administered at the maximum tolerated dose (MTD), requires a break period to avoid adverse toxic events. The breaks sometimes allow tumor regrowth. On the other hand, low dose metronomic chemotherapy (LDM) means frequent low dose administration of the cytotoxic agent without breaks. Here, we investigated the therapeutic efficacies of LDM using S-1, a novel 5-FU based drug, and with Vandetanib (VEGFR-2 and EGFR inhibitors), an anti-angiogenic agent. [Materials and Methods] The inhibitory effects of 5-FU on the proliferation of HUVEC and human hepatoma cell lines were evaluated in vitro. We compared the anti-tumor effect, toxicity and survival in the following 6 groups; MTD and LDM using S-1, Vandetanib (25 mg/kg/day), S-1 MTD + Vandetanib and S-1 LDM + Vandetanib. In the S-1 MTD group, mice were administered orally at 15 mg/kg/day for 7 days followed by a break for 7 days. In the S-1 LDM group, S-1 of 5mg/kg/day was consecutively administered once a day without a break. In the subcutaneous tumor model, tumor volumes and toxicities were evaluated. In the liver xenograft model, tumor volumes and survival were evaluated. Micro vessel density (MVD) in tumor tissues was evaluated by immunohistochemistry using an anti CD-31 antibody. The expression of VEGF and thrombospondin-1 (an endogeneous anti-angiogenic factor: TSP-1) was analysed by Western blot. [Results] Cell proliferation of HUVEC and hepatoma cell lines was inhibited at apparently lower doses in the metronomic schedule compared with the MTD schedule. In the subcutaneous tumor model, S-1 LDM significantly inhibited tumor growth (control: 4,810 ± 1,440 mm3, S-1 MTD: 3,212 ± 1,364 mm3, S-1 LDM: 1,927 ± 652 mm3) and its effect was enhanced by the combination with Vandetanib (LDM S-1 + Vandetanib: 1,383 ± 697 mm3). In toxicities, S-1 MTD caused severe body weight loss, and myelo-suppression. On the other hand, S-1 LDM did not show any toxicities. In the liver xenograft model, S-1 LDM and the combination therapy with Vandetanib significantly inhibited tumor growth and S-1 LDM apparently prolonged survival (control: 28.9 ± 6.4 days, S-1 MTD: 29.6 ± 3.9 days, S-1 LDM: 34.3 ± 3.9 days). S-1 LDM + Vandetanib showed the most prolonged survival (49.6 ± 11.5 days). MVDs in the tumor of control, S-1 MTD, S-1 LDM, S-1 LDM + Vandetanib were 41.1 ± 9.2 /field, 35.8 ± 5.5 /field, 17.2 ± 4.1 /field, and 8.2 ± 1.6 /field, respectively. The expression of VEGF and TSP-1 was strongly upregulated in S-1 LDM group and S-1 LDM + Vandetanib group. [Conclusion] S-1 LDM significantly suppressed tumor growth and prolonged survival without severe toxicities. These anti-tumor effects were enhanced by the combination therapy with Vandetanib. These results indicate that S-1 LDM with an anti-angiogenic agent will become a novel therapeutic strategy for human hepatocellular carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1389.

Novel chemoembolization using calcium‐phosphate ceramic microsphere incorporating TNP‐470, an anti‐angiogenic agent

The purpose of the present study was to develop a new method of chemoembolization to improve the therapeutic effectiveness and safety profile of cancer treatment. A chemoembolization approach was designed for human solid tumors using resorbable calcium-phosphate ceramic microspheres loaded with an agent anti-angiogenic to tumor vasculature in vivo. The human uterine sarcoma cell line FU-MMT-3 was used in this study because this tumor is aggressive and also exhibits a poor response to radiotherapy or any chemotherapy currently used. The calcium-phosphate ceramic microspheres loaded with TNP-470, an anti-angiogenic agent, were injected into FU-MMT-3 xenografts in nude mice three times per week for 8 weeks. The treatment using TNP-470-loaded microspheres suppressed tumor growth, compared to treatment with TNP-470 alone, microspheres alone, and the control. The mean tumor weight after treatment using TNP-470-loaded microspheres was significantly lower than that after treatment with microspheres alone. These ceramic microspheres were remarkably embolized in tumor microvessels as well as in the feeding arteries and a significant reduction of intratumoral vascularity was also demonstrated following treatment with TNP-470-loaded microspheres. Severe loss of body weight was not observed in any mice treated with the TNP-470-loaded microspheres, compared to treatment with TNP-470 alone. These results suggest that targeting tumor vasculature in human uterine sarcoma using calcium-phosphate microspheres might be more effective and safer than the treatment that employs anti-angiogenic agent alone. This new chemoembolization method incorporating an anti-angiogenic agent may contribute to the effective treatment of locally advanced or recurrent solid tumors.

Mice lacking adenylyl cyclase‐5 cope badly with repeated restraint stress

Physiological responses to acute stress proceed with the activation of the hypothalamus-pituitary-adrenal gland (HPA) system. Many brain regions are known to modulate the HPA axis activation in stress responses, but the detailed neural circuits and signaling system in the upstream of the HPA axis have to be explored further. Type 5 adenylyl cyclase (AC5) is highly concentrated in the dorsal striatum and nucleus accumbens, which are implicated in reward and stress-related behavior. AC5(-/-) mice exposed to daily 2-hr restraint stress for only 3-5 days showed poor stress-coping responses, including severe body weight loss, poor coat condition, respiratory difficulties, and freezing behavior. Plasma corticosterone levels during 2-hr stress sessions increased in AC5(-/-) mice compared with those of AC5(+/+) mice. However, neither the corticosterone receptor antagonist RU486 nor the CRH receptor antagonist NBI27914 blocked their poor stress coping, whereas the administration of the GABA(A) receptor allosteric modulator diazepam or the D1 dopamine receptor antagonist SCH23390 prior to restraint stress sessions changed their stress-coping response to the stressed AC5(+/+) mouse level. Stress-triggered c-Fos expression was completely blunted in the dorsal striatum of AC5(-/-). These results suggest that the AC5-associated signal system and neural network are involved in the regulation of anxiety and stress-coping response.