Dietary copper supplementation restores ß-cell function of Cohen diabetic rats: a link between mitochondrial function and glucose-stimulated insulin secretion.

ß-Cell mitochondrial dysfunction as well as proinflammatory cytokines have been suggested to contribute to reduced glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. We recently demonstrated that Cohen diabetic sensitive (CDs) rats fed a high-sucrose, low-copper diet (HSD) developed hyperglycemia and reduced GSIS in association with peri-islet infiltration of fat and interleukin (IL)-1ß-expressing macrophages, whereas CD resistant (CDr) rats remained normoglycemic on HSD. We examined: 1) the correlation between copper concentration in the HSD and progression, prevention, and reversion of hyperglycemia in CDs rats, 2) the relationship between activity of the copper-dependent, respiratory-chain enzyme cytochrome c oxidase (COX), infiltration of fat, IL-1ß-expressing macrophages, and defective GSIS in hyperglycemic CDs rats. CDs and CDr rats were fed HSD or copper-supplemented HSD before and during hyperglycemia development. Blood glucose and insulin concentrations were measured during glucose tolerance tests. Macrophage infiltration and IL-1ß expression were evaluated in pancreatic sections by electron-microscopy and immunostaining. COX activity was measured in pancreatic sections and isolated islets. In CDs rats fed HSD, GSIS and islet COX activity decreased, while blood glucose and infiltration of fat and IL-1ß-expressing macrophages increased with time on HSD (P < 0.01 vs. CDr-HSD rats, all parameters, respectively). CDs rats maintained on copper-supplemented HSD did not develop hyperglycemia, and in hyperglycemic CDs rats, copper supplementation restored GSIS and COX activity, reversed hyperglycemia and infiltration of fat and IL-1ß-expressing macrophages (P < 0.01 vs. hyperglycemic CDs-HSD rats, all parameters, respectively). We provide novel evidence for a critical role of low dietary copper in diminished GSIS of susceptible CDs rats involving the combined consequence of reduced islet COX activity and pancreatic low-grade inflammation.

Steps toward mapping the human vasculature by phage display.

The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.

Targeted anti-vascular endothelial growth factor receptor-2 therapy leads to short-term and long-term impairment of vascular function and increase in tumor hypoxia.

Because antiangiogenic therapies inhibit the growth of new tumor-associated blood vessels, as well as prune newly formed vasculature, they would be expected to reduce the supply of oxygen and thus increase tumor hypoxia. However, it is not clear if antiangiogenic treatments lead only to consistent and sustained increases in hypoxia, or transient decreases in tumor hypoxia along with periods of increased hypoxia. We undertook a detailed analysis of an orthotopically transplanted human breast carcinoma (MDA-MB-231) over a 3-week treatment period using DC101, an anti-vascular endothelial growth factor receptor 2 antibody. We observed consistent reductions in microvascular density, blood flow (measured by high-frequency micro-ultrasound), and perfusion. These effects resulted in an increase in the hypoxic tumor fraction, measured with an exogenous marker, pimonidazole, concurrent with an elevation in hypoxia-inducible factor-1alpha expression, an endogenous marker. The increase in tumor hypoxia was evident within 5 days and remained so throughout the entire course of treatment. Vascular perfusion and flow were impaired at days 2, 5, 7, 8, 14, and 21 after the first injection, but not at 4 hours. A modest increase in the vessel maturation index was detected after the 3-week treatment period, but this was not accompanied by an improvement in vascular function. These results suggest that sustained hypoxia and impairment of vascular function can be two consistent consequences of antiangiogenic drug treatment. The implications of the results are discussed, particularly with respect to how they relate to different theories for the counterintuitive chemosensitizing effects of antiangiogenic drugs, even when hypoxia is increased.

Low-dose metronomic combined with intermittent bolus-dose cyclophosphamide is an effective long-term chemotherapy treatment strategy.

Metronomic chemotherapy refers to the close, regular administration of comparatively low doses of cytotoxic drugs, with minimal or no drug-free breaks, over prolonged periods. It is thought to have an antiangiogenic basis. However, whereas surprisingly durable and potent tumor responses have been observed in a number of preclinical tumor models, relapses usually eventually occur using this type of treatment strategy. We therefore decided to test modified metronomic chemotherapy regimens that might significantly delay such relapses, but still maintain modest and acceptable toxicity profiles. Here, we show that repeated administration of bolus doses (BDs) of cyclophosphamide every 3 or 6 weeks, combined with a daily oral low-dose metronomic (LDM) regimen (20 mg/kg/d cyclophosphamide), improves efficacy and significantly delays progression of transplanted PC-3 human prostate cancer xenografts, syngeneic transplanted EMT-6 breast tumors, and "spontaneous" murine erythroleukemia. Efficacy was superior whereas toxicity was mild and comparable to the LDM regimen, the latter assessed by body weight, neutrophil, lymphocyte, and total white blood counts. Antiangiogenic activity, measured by reduction in circulating endothelial precursor cells, revealed that the greatest degree of suppression occurred using the combination treatment. Overall, our results indicate that the administration of intermittent BD combined with chronic oral LDM cyclophosphamide is a potent treatment regimen for controlling tumor growth, which has a low toxicity profile, over prolonged periods of time.

Design and validation of a bifunctional ligand display system for receptor targeting.

Here we developed a bacteriophage display particle designed to serve as a bifunctional entity that can target tumors while delivering an agent. We engineered a chimera phage vector containing a pIII-displayed alphav integrins-targeting moiety and a pVIII-displayed streptavidin binding adaptor moiety. By using the chimeric phage particle, targeting of alphav integrins on cells in culture and tumor-related blood vessels was shown through different applications, including luminescent quantum dots localization, surface plasmon resonance-based binding detection, and an in vivo tumor model. The strategy validated here will accelerate the discovery and characterization of receptor-ligand binding events in high throughput, and cell-specific delivery of diagnostics or therapeutics to organs of choice without the need for chemical conjugation.

Reactive antibodies against bacillus Calmette-Guerin heat-shock protein-65 potentially predict the outcome of immunotherapy for high-grade transitional cell carcinoma of the bladder.

BACKGROUND: Intravesical immunotherapy with Mycobacterium bovis (M. bovis) bacillus Calmette-Guerin (BCG) is the current standard of care against superficial, high-grade transitional cell carcinoma (TCC) of the urinary bladder (carcinoma in situ and pathologic T1, grade 3 disease). However, individual patient outcome is barely predictable because of the lack of serum markers. Consequently, progression to muscle-invasive bladder cancer and critical delay of treatments (such as neoadjuvant combination chemotherapy and/or radical cystectomy) often occur. The objectives of this study were to identify a marker for measuring the BCG-induced immune response and to predict the outcomes and potential improvements of BCG immunotherapy. METHODS: Because host immunoresponse mediates BCG activity, the authors screened a combinatorial random peptide library on the circulating pool of immunoglobulins (Igs) purified from an index patient after successful BCG immunotherapy to identify the corresponding target antigen(s). RESULTS: An immunogenic peptide motif was selected, isolated, and validated from M. bovis BCG heat-shock protein 65 (HSP-65) as a dominant epitope of the humoral response to treatment. Increasing IgA and IgG anti-HSP-65 titers specifically predicted a positive patient outcome in a cohort of patients with bladder cancer relative to several cohorts of control patients. CONCLUSIONS: The current results indicated that antibody production against M. bovis BCG HSP-65 can serve as a serologic marker for the predictive outcome of BCG immunotherapy. Subsequent studies will determine the value of this candidate marker to modify BCG-based treatment for individual patients with bladder cancer.

The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells.

The stromal compartments of hematopoietic organs (eg, spleen) are known to influence the viability and growth of diseased hematopoietic progenitors. Here we have used Friend murine leukemia virus (F-MuLV)-induced erythroleukemia to investigate factors of the splenic microenvironment that may make it fertile for the expansion and survival of malignant erythroblasts. We found that splenectomized, erythroleukemic mice exhibited extended survival compared with age-matched sham controls. In vitro, the proliferation of primary erythroleukemic cells cocultured with leukemic-derived splenic adherent cells or their conditioned media was found to be significantly higher than that observed in cocultures with healthy-derived adherent splenic cells. Cytokine protein arrays revealed that F-MuLV-infected splenocytes secreted elevated levels of interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), macrophage chemoattractant protein-5 (MCP-5), soluble tumor necrosis factor receptor-1 (sTNFR1), IL-12p70, tumor necrosis factor-alpha (TNF-alpha), and IL-2 over normal splenocytes. Medium supplemented with both VEGF-A and MCP-5 could sustain proliferation of primary erythroleukemic cells in vitro, and significant proliferative suppression was observed upon addition of neutralizing antibodies to either of these factors. Furthermore, in vivo administration of a neutralizing antibody to VEGF-A extended survival times of erythroleukemic mice in comparison with controls. These findings suggest that VEGF-A and MCP-5 are potentially pivotal paracrine mediators occurring within the diseased splenic microenvironment capable of promoting disease acceleration and expansion of erythroleukemic blasts.

Maternal autoimmune diseases and immunologically induced embryonic and fetoplacental damage.

BACKGROUND: Autoimmune diseases are a group of illnesses in which autoantibodies are produced against various organs, presenting with a variety of clinical symptoms. In this review, we discuss the different aspects of autoimmune diseases in pregnancy. We also describe experimental models that help to understand the etiology and pathogenesis of the effects of this maternal disease on the developing embryo, fetus and placenta. METHODS: The possible direct effects of sera or IgG obtained from women with systemic lupus erythematosus/antiphospholipid syndrome (SLE/APS) and recurrent pregnancy loss (RPL) were examined on cultured 10.5- and 11.5-day-old rat embryos and on cultured human placental explants, as compared to sera from healthy women or synthetic medium that were used as controls. In addition, we examined the effects of the sera obtained from these women after successful treatment that allowed the birth of normal infants. RESULTS: We observed increased embryonic death and anomalies in embryos cultured on sera and IgG from SLE/APS women. Similarly, when human placental explants were cultured on these sera, trophoblastic cell growth was reduced and apoptotic rate was increased. Successful treatment also reduced the damage caused by the sera from these women in the cultured embryos and placentas. CONCLUSIONS: Our results, and the cited studies, point to the important role of the placental damage in the etiology of RPL associated with SLE/APS. Animal models, both in vivo and in vitro, as well as cultured early human placental explants can be used successfully to understand some of the pathogenic aspects of SLE/APS and RPL.

Targeting urothelium: ex vivo assay standardization and selection of internalizing ligands.

PURPOSE: With the goal of targeting the human bladder using phage display technology we designed and tested a tissue binding assay on intact urothelium ex vivo. This approach may form the molecular basis for clinical development of peptide or peptidomimetic guided intravesical compounds. MATERIALS AND METHODS: We screened 2 phage display random peptide libraries on human urothelium. Select peptides were tested for their binding ability to human urothelium, 2 human transitional cell carcinoma cell lines and a nontransitional cell carcinoma cell line. Next we standardized an ex vivo binding assay, validated binding of selected phage to whole urothelium, and evaluated whether receptor mediated internalization into urothelium derived cells occurred. Finally we tested if the presence of the glycosaminoglycan layer had any effect on the binding of the urothelium targeted phage. RESULTS: Phage selected and recovered in the screening were isolated and sequenced. Displayed peptide sequences were searched against online protein databases. Five classes of peptide motifs were characterized based on their ability to bind to normal urothelium but not to control cell lines. Remarkable consistency and reproducibility were observed in the ex vivo binding assays. Two classes of peptide motifs sharing the sequence Ile/Leu-Ser-Gly-Leu bound to normal urothelium and to 2 transitional cell carcinoma cells but not to nontransitional cell carcinoma cells in a glycosaminoglycan independent manner and mediated internalization into cells of urothelial origin. CONCLUSIONS: We introduce a strategy for screening combinatorial peptide libraries on bladder mucosa, a standard model for ex vivo intact urothelium binding assays and a panel of urothelium binding peptides that may be suitable for translation into targeted intravesical therapy applications.