Angiostatin enhances B7.1-mediated cancer immunotherapy independently of effects on vascular endothelial growth factor expression
Tumors must develop an adequate vascular network to meet their increasing demands for nutrition and oxygen. Angiostatin, a multiple kringle (1–4)–containing fragment of plasminogen, is an effective natural inhibitor of tumor angiogenesis. Here we show that gene transfer of angiostatin into small (0.1 cm in diameter) solid EL-4 lymphomas established in syngeneic C57BL/6 mice led to reduced tumor angiogenesis and weak inhibition of tumor growth. In contrast, when angiostatin gene therapy was preceded by in situ gene transfer of the T-cell costimulator B7.1, large (0.4 cm in diameter) tumors were rapidly and completely eradicated, whereas B7.1 and angiostatin monotherapies were ineffective. Combined gene transfer of B7.1 and angiostatin generated potent systemic antitumor immunity that was effective in eradicating a systemic challenge of 107 EL-4 cells. Gene transfer of angiostatin expression plasmids led to overexpression of angiostatin in tumors, increased apoptosis of tumor cells, and decreased density of tumor blood vessels, which may allow the immune system to overcome tumor immune resistance. The latter effects were not the result of a decrease in vascular endothelial growth factor expression, as tumoral vascular endothelial growth factor expression increased slightly after angiostatin gene transfer, presumably in response to increasing hypoxia. These results suggest that combining immunogene therapy with a vascular attack by angiostatin is a particularly effective approach for eliciting antitumor immunity. Cancer Gene Therapy (2001) 8, 719–727

title: Beta7 integrins contribute to skin graft rejection.
abstract: Because integrins alpha4beta7 and alphaEbeta7 contribute to epidermotropism of T-cells during skin inflammation, we sought to study their role in skin allograft rejection.



Wild-type (WT) (beta7+/+) and beta7 gene knockout (beta7-/-) C57BL/6 (H-2(b)) mice and SJL/J (H-2(s)) mice served as donors and recipients of allogeneic skin grafts. An anti-integrin beta7 subunit mAb (FIB504.64) was used to treat WT beta7+/+ C57BL/6 recipients of skin grafts from SJL/J mice.



WT C57BL/6 recipients acutely rejected skin from SJL/J mice in 13 days. In contrast, the survival of SJL/J skin on either beta7-/- gene knockout or WT C57BL/6 recipients treated with anti-beta7 subunit mAb, was prolonged by 6 to 7 additional days (P<0.01). The survival of skin allografts from either beta7-/- or beta7+/+ C57BL/6 mice received by SJL/J recipients was not prolonged (P >0.05).



Beta7 integrins contribute to skin graft rejection, in accord with their role in mediating the epidermotropism of T-cells during skin inflammation.

title: Intramuscular delivery of antiangiogenic genes suppresses secondary metastases after removal of primary tumors
abstract: The success of surgery to remove primary tumors can be compromised by the subsequent outgrowth of metastases. It is recognized that primary tumors secrete antiangiogenic factors that suppress the outgrowth of their daughter metastases. In accord we show here that surgical removal of primary EL-4 lymphomas led to a marked decrease in the levels of circulating angiostatin and endostatin, and promoted the growth of distant nodular tumors. Expression vectors encoding angiostatin and endostatin, formulated with poly-N-vinyl pyrrolidone (PVP), were injected into the tibialis and gastrocnemia muscles, leading to expression of angiostatin and endostatin in muscle fibers. High levels of biologically active exogenous proteins were secreted into the circulation. Intramuscular gene therapy with angiostatin and endostatin plasmids significantly inhibited tumor vascularity and induced tumor cell apoptosis, and thereby suppressed the growth of secondary subcutaneous and disseminated metastatic tumors in the lung and liver. Simultaneous intramuscular delivery of both angiostatin and endostatin plasmids significantly prolonged the survival of mice after removal of primary tumors. These results suggest that intramuscular gene transfer of angiostatin and endostatin might serve as a prophylactic cancer-prevention strategy to combat the recurrence of cancer after surgical resection of primary tumors.

title: Anti-angiogenic therapy subsequent to adeno-associated-virus-mediated immunotherapy eradicates lymphomas that disseminate to the liver.
abstract: Liver cancer has a very poor prognosis and lacks effective therapy. We have previously demonstrated that intraportal injection of adeno-associated-viral (AAV) particles that express angiostatin lead to long-term expression of angiostatin capable of suppressing the outgrowth of EL-4 tumors in the liver. Here we combine AAV-mediated angiostatin therapy with immunotherapy by employing an AAV vector encoding the T-cell costimulator B7.1. Incubation of EL-4 cells with AAV-B7.1 viruses resulted in the rapid expression of B7.1 on the surface of 80% of EL-4 cells. Mice that were vaccinated with B7.1-engineered tumor cells rejected the tumor cells and resisted a secondary challenge with unmodified parental cells. Splenocytes from the vaccinated mice were highly cytotoxic towards parental EL-4 cells in vitro. However, the vaccinated mice failed to resist the challenge of a heavy burden of EL-4 cells. Intraportal injection of AAV particles that express angiostatin into mice that had been vaccinated 1 month earlier with B7.1-engineered tumor cells protected mice against the challenge of a heavy burden of EL-4 cells and eradicated tumors that had disseminated to the liver. The combinational therapy increased the survival rate of mice with advanced liver cancer. These encouraging results warrant investigation of the employment of anti-angiogenic therapy subsequent to cancer immunotherapy for targeting unresectable disseminated liver metastases.

title: Antisense HIF-1α prevents acquired tumor resistance to angiostatin gene therapy
abstract: Angiostatin is a naturally occurring inhibitor of angiogenesis that is being developed as a drug to fight cancer. In this study we reveal that EL-4 tumors established in mice rapidly develop resistance to angiostatin gene therapy by upregulating hypoxia-inducible pathways. Angiostatin initially delayed tumor growth for 6 days by reducing blood vessel density. However, tumors quickly responded by upregulating the production of hypoxia-inducible factor-1α (HIF-1α) and its effector vascular endothelial growth factor (VEGF) in response to increasing tumor hypoxia, leading to restored angiogenesis and rapid tumor growth. Theoretically, blockade of HIF-1 should prevent resistance to anti-angiogenic therapy by preventing a tumor from responding to induced hypoxia. Antisense HIF-1α inhibited the expression of HIF-1α and of the HIF-1 effectors VEGF, glucose transporter-1 and lactate dehydrogenase. As a monotherapy, it was effective in eradicating small 0.1 cm diameter tumors, but only delayed the growth of large 0.4 cm diameter tumors. In contrast, timed injection of a combination of angiostatin and antisense HIF-1α plasmids completely eradicated large EL-4 tumors within 2 weeks, and prevented upregulation of hypoxia-inducible pathways induced by angiostatin. The data indicate that blocking hypoxia-inducible pathways by antisense HIF-1α can circumvent hypoxia-induced drug resistance and thereby augment the efficacy of anti-angiogenic therapies.

title: Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1α eradicates gliomas
abstract: The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.

title: Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis.
abstract: Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.

title: In-column “click” preparation of hydrophobic organic monolithic stationary phases for protein separation
abstract: AbstractTwo types of macroporous organic polymer monoliths based on glycidyl methacrylate (GMA), 4-vinylbenzyl chloride (VBC) and divinylbenzene (DVB) were prepared inside stainless-steel tubes. Azide functionalities were firstly introduced on the surfaces of poly(GMA-co-DVB) and poly(VBC-co-DVB) monoliths to provide reactive sites for click chemistry. With the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths was developed. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by the scanning electron microscopy, mercury intrusion porosimeter, Fourier transform infrared spectroscopy, and elemental analyses, respectively. The chromatographic performances of the “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min.
 FigureWith the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition (CuAAC), an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths based on poly(GMA-co-DVB)/poly(VBC-co-DVB) was developed. The “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min

title: Interruption of nuclear localization of ATBF1 during the histopathologic progression of head and neck squamous cell carcinoma.
abstract: The AT-motif binding factor 1 (ATBF1) gene is frequently altered at the genetic level in several types of cancer, but its protein expression and subcellular localization have not been well studied in human cancers, including head and neck squamous cell carcinomas (HNSCCs).



ATBF1 expression and localization were examined in 5 cell lines and 197 clinical specimens of HNSCC, and correlated with pathologic and clinical characteristics.



ATBF1 was predominantly localized in the nucleus of hyperplastic squamous epithelium. Whereas nuclear ATBF1 dramatically decreased in invasive tumors (p = .0012), cytoplasmic ATBF1 levels progressively increased from dysplasia to invasive tumors (p < .0001), and the increase correlated with poor survival. Reduced nuclear ATBF1 level was also detected in HNSCC cell lines.



Nuclear localization of ATBF1 is frequently interrupted in HNSCC, and the interruption is significantly associated with the progression of HNSCC. The cytoplasmic ATBF1 level could be useful for predicting patient survival.

title: A methodological framework of preparing economic evidence for selection of medicines in the Chinese setting.
abstract: Medicines are becoming a major component of health expenditure in China. Selection of effective and cost-effective medicines represents an important effort to improve medicines use. A guideline on cost-effectiveness studies has been available in China. This guideline, however, fails to be a practical tool to prepare and critically appraise economic evidence. This article discusses, in the Chinese context, the approach to integrating economic component into the medicines selection, and elaborates the methods of producing economic evidence, including conducing economic reviews and primary economic studies.

title: Subgroup Analysis of Trials Is Rarely Easy (SATIRE): a study protocol for a systematic review to characterize the analysis, reporting, and claim of subgroup effects in randomized trials
abstract: BackgroundSubgroup analyses in randomized trials examine whether effects of interventions differ between subgroups of study populations according to characteristics of patients or interventions. However, findings from subgroup analyses may be misleading, potentially resulting in suboptimal clinical and health decision making. Few studies have investigated the reporting and conduct of subgroup analyses and a number of important questions remain unanswered. The objectives of this study are: 1) to describe the reporting of subgroup analyses and claims of subgroup effects in randomized controlled trials, 2) to assess study characteristics associated with reporting of subgroup analyses and with claims of subgroup effects, and 3) to examine the analysis, and interpretation of subgroup effects for each study's primary outcome.MethodsWe will conduct a systematic review of 464 randomized controlled human trials published in 2007 in the 118 Core Clinical Journals defined by the National Library of Medicine. We will randomly select journal articles, stratified in a 1:1 ratio by higher impact versus lower impact journals. According to 2007 ISI total citations, we consider the New England Journal of Medicine, JAMA, Lancet, Annals of Internal Medicine, and BMJ as higher impact journals. Teams of two reviewers will independently screen full texts of reports for eligibility, and abstract data, using standardized, pilot-tested extraction forms. We will conduct univariable and multivariable logistic regression analyses to examine the association of pre-specified study characteristics with reporting of subgroup analyses and with claims of subgroup effects for the primary and any other outcomes.DiscussionA clear understanding of subgroup analyses, as currently conducted and reported in published randomized controlled trials, will reveal both strengths and weaknesses of this practice. Our findings will contribute to a set of recommendations to optimize the conduct and reporting of subgroup analyses, and claim and interpretation of subgroup effects in randomized trials.