Maté Tea Inhibits In Vitro Pancreatic Lipase Activity and Has Hypolipidemic Effect on High‐fat Diet‐induced Obese Mice

The inhibitory effects of maté tea (MT), a beverage produced with leaves from Ilex paraguariensis, in vitro lipase activity and on obesity in obese mice models were examined. For the in vitro experiment, porcine and human pancreatic lipase (PL) activities were determined by measuring the rate of release of oleic acid from hydrolysis of olive oil emulsified with taurocholate, phospholipids, gum arabic, or polyvinyl alcohol. For the in vivo experiments, animals were fed with a standard diet (SD, n = 10) or high‐fat diet (HFD, n = 30) for 16 weeks. After the first 8 weeks on the HFD, the animals were treated with 1 and 2 g/kg of body weight of MT. The time course of the body weight and obesity‐related biochemical parameters were evaluated. The results showed that MT inhibited both porcine and human PL (half‐maximal inhibitory concentration = 1.5 mg MT/ml) and induced a strong inhibition of the porcine lipase activity in the hydrolysis of substrate emulsified with taurocholate + phosphatidylcholine (PC) (83 ± 3.8%) or PC alone (62 ± 4.3%). MT suppressed the increases in body weight (P < 0.05) and decreased the serum triglycerides and low‐density lipoprotein (LDL)‐cholesterol concentrations at both doses (from 190.3 ± 5.7 to 135.0 ± 8.9 mg/dl, from 189.1 ± 7.3 to 129.3 ± 17.6 mg/dl; P < 0.05, respectively) after they had been increased by the HFD. The liver lipid content was also decreased by the diet containing MT (from 132.6 ± 3.9 to 95.6 ± 6.1 mg/g of tissue; P < 0.05). These results suggest that MT could be a potentially therapeutic alternative in the treatment of obesity caused by a HFD.     

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.     

Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity

Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-gamma. T cells conditioned with the B7-H1-blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.     

Genetically engineered intracellular single-chain antibodies in gene therapy

The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins.     

Macrophage-derived dendritic cells have strong Th1-polarizing potential mediated by beta-chemokines rather than IL-12

Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MPhis) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MPhi-derived DCs (PhiDCs). Monocytes were cultured in MPhi-CSF (M-CSF) to produce MPhis, which were then differentiated into DCs following culture with GM-CSF plus IL-4. PhiDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, PhiDCs elicited up to 9-fold more IFN-gamma from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not PhiDC IL-12 significantly inhibited T cell IFN-gamma induction. PhiDCs produced up to 12-fold more beta-chemokines (macrophage-inflammatory protein-1alpha, -1beta, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-gamma induction by PhiDCs significantly greater than by MDDCs. Thus DCs differentiating from MPhis induce T cell IFN-gamma through beta-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-gamma induction through chemokines by DCs.     

Identification of sites in adenovirus hexon for foreign peptide incorporation

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.